Detergent Compositions Containing Bacillus Sp. Mannanase And Methods Of Use Thereof

Abstract

The present compositions and methods relate to an endo-B-mannanase cloned from a Bacillus sp., polynucleotides encoding the endo-B-mannanase, and methods of use thereof. Formulations containing the endo-.beta.-mannanase are highly suitable for use as detergents.

Description

PRIORITY

[0001] The present application claims priority to International Application No. PCT/CN2011/073559, filed on Apr. 29, 2011, which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] The present compositions and methods relate to an endo-.beta.-mannanase cloned from a Bacillus sp., polynucleotides encoding the endo-.beta.-mannanase, and methods of use thereof. Formulations containing the endo-.beta.-mannanase are highly suitable for use as detergents.

BACKGROUND

[0003] Current laundry detergent and fabric care compositions include a complex combination of active ingredients such as surfactants, enzymes (protease, amylase, mannanase, and/or cellulase), bleaching agents, a builder system, suds suppressors, soil-suspending agents, soil-release agents, optical brighteners, softening agents, dispersants, dye transfer inhibition compounds, abrasives, bactericides, and perfumes.

[0004] Mannanase enzymes, including endo-.beta.-mannanases, have been employed in detergent cleaning compositions for the removal of gum stains by hydrolyzing mannans. A variety of mannans are found in nature. These include linear mannan, glucomannan, galactomannan, and glucogalactomannan. In each case, the polysaccharide contains a .beta.-1,4-linked backbone of mannose residues that may be substituted up to 33% with glucose residues (Yeoman et al., Adv Appl Microbiol, Elsivier). In galactomannans or glucogalactomannnans, galactose residues are linked in alpha-1,6-linkages to the mannan backbone (Moreira and Filho, Appl Microbiol Biotechnol, 79:165, 2008). Therefore, hydrolysis of mannan to its component sugars requires endo-1,4-.beta.-mannanases that hydrolyze the backbone linkages to generate short chain manno-oligosaccharides that are further degraded to monosaccharides by 1,4-.beta.-mannosidases.

[0005] However, enzymes are often inhibited by surfactants and other components present in cleaning compositions, which interferes with their ability to remove stains. For instance, proteases present in laundry detergents may degrade mannanases before the removal of a gum stain occurs. In addition, mannanases may have a limited pH and/or temperature range at which they are active, which may make them unsuitable for certain formulations and washing conditions. Accordingly, the need exists for endo-.beta.-mannanases that retain activity in the harsh environment of cleaning compositions.

SUMMARY

[0006] The present compositions and methods relate to endo-.beta.-mannanasel cloned from Bacillus sp. SWT81 (Bsp Man4). Formulations containing the endo-.beta.-mannanase are highly suitable for use in detergents, food or feed.

[0007] In particular the present disclosure provides recombinant polypeptides comprising a catalytic domain of an endo-.beta.-mannanase, wherein the catalytic domain is at least 85% (85%, 86%, 87%, 88%, 89%, 90, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO:9. The present disclosure also provides recombinant polypeptides comprising a mature form of an endo-.beta.-mannanase, wherein the mature form is at least 80% (80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO:8. In some embodiments, the polypeptide has measurable mannanase activity in the presence of detergent. In some embodiments, the polypeptide has measurable mannanase activity in the presence of a protease. In some embodiments, the polypeptide and the protease are both present at from about 0.1 to about 10.0 ppm. In some embodiments, the polypeptide retains greater than 70% mannanase activity at pH values of between 6 and 8.5. In some embodiments, the polypeptide has a pH optimum of about 6.5. In some embodiments, the polypeptide retains greater than 70% mannanase activity at a temperature range from 55.degree. C. to 65.degree. C. In some embodiments, the polypeptide has a temperature optimum of about 60.degree. C. In some embodiments, the polypeptide is capable of hydrolyzing a substrate selected from the group consisting of chocolate ice cream, guar gum, locust bean gum, and combinations thereof. In some embodiments, wherein the amino acid sequence is at least 95% identical to one of the group consisting of SEQ ID NOS:6-14 and 30-49. In some embodiments, the polypeptide further comprises an amino-terminal extension of Ala-Gly-Lys. In some embodiments, the polypeptide further comprises a native or non-native signal peptide. In some embodiments, the polypeptide further comprises at least one carbohydrate-binding module. In other embodiments, the polypeptide does not comprise a carbohydrate-binding module.

[0008] Also provided by the present disclosure are detergent compositions comprising at least one recombinant polypeptide of the preceding paragraph. In some embodiments, the composition further comprises a surfactant. In some embodiments, the surfactant is selected from the group consisting of sodium dodecylbenzene sulfonate, sodium hydrogenated cocoate, sodium laureth sulfate, C12-14 pareth-7, C12-15 pareth-7, sodium C12-15 pareth sulfate, C14-15 pareth-4, and combinations thereof. In some preferred embodiments, the surfactant is an ionic surfactant. In some embodiments, the ionic surfactant is selected from the group consisting of an anionic surfactant, a cationic surfactant, a zwitterionic surfactant, and a combination thereof. In some preferred embodiments, the composition further comprises an enzyme selected from the group consisting proteases, proteases, peroxidases, cellulases, beta-glucanases, hemicellulases, lipases, acyl transferases, phospholipases, esterases, laccases, catalases, aryl esterases, amylases, alpha-amylases, glucoamylases, cutinases, pectinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenases, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, other endo-.beta.-mannanases, exo-.beta.-mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, and combinations thereof. In some embodiments, the combination comprises a protease and an amylase. In some embodiments, the detergent is selected from the group consisting of a laundry detergent, a fabric softening detergent, a dishwashing detergent, and a hard-surface cleaning detergent. In some embodiments, the detergent is in a form selected from the group consisting of a liquid, a powder, a granulated solid, and a tablet. In addition the present disclosure provides methods for hydrolyzing a mannan substrate present in a soil or stain on a surface, comprising: contacting the surface with the detergent composition to produce a clean surface. Also provided are methods of textile cleaning comprising: contacting a soiled textile with the detergent composition to produce a clean textile.

[0009] Moreover, the present disclosure provides isolated nucleic acids encoding the recombinant polypeptide of the preceding paragraphs. Also provided are expression vectors comprising the isolated nucleic acid in operable combination to a regulatory sequence. Additionally, host cells comprising the expression vector are provided. In some embodiments, the host cell is a bacterial cell or a fungal cell. The present disclosure further provides methods of producing an endo-.beta.-mannanase, comprising: culturing the host cell in a culture medium, under suitable conditions to produce a culture comprising the endo-.beta.-mannanase. In some embodiments, the methods further comprise removing the host cells from the culture by centrifugation, and removing debris of less than 10 kDa by filtration to produce an endo-.beta.-mannanase-enriched supernatant. The present disclosure further provides methods for hydrolyzing a polysaccharide, comprising: contacting a polysaccharide comprising mannose with the supernatant to produce oligosaccharides comprising mannose. In some embodiments, the polysaccharide is selected from the group consisting of mannan, glucomannan, galactomannan, galactoglucomannan, and combinations thereof.

[0010] Also provided by the present disclosure are food or feed compositions having at least one recombinant polypeptide as described above, methods of preparing these compositions and uses of these compositions. This includes animal and/or human food or feed, which also includes fermented beverages.

[0011] These and other aspects of Bsp Man4 compositions and methods will be apparent from the following description.

DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 provides a plasmid map of pZQ186 (aprE-Bsp Man4).

[0013] FIG. 2A shows the pH profile of Bsp Man4. FIG. 2B shows the pH profile for a benchmark endo-.beta.-mannanase (Mannastar.TM.).

[0014] FIG. 3A shows the temperature profile of Bsp Man4. FIG. 3B shows the temperature profile of a benchmark endo-.beta.-mannanase (Mannastar.TM.).

[0015] FIG. 4A shows the mannanase activity of various forms of Bsp Man4 at 30.degree. C., for 30 min at pH 8.2. FIG. 4B shows the mannanase activity of various forms of Bsp Man4 at 50.degree. C., for 10 min at pH 5.

[0016] FIG. 5A shows the cleaning performance of Bsp Man4 in Small & Mighty liquid detergent. FIG. 5B shows the cleaning performance of Bsp Man4 in OMO Color powder detergent.

[0017] FIG. 6A shows the cleaning performance of various forms of Bsp Man4 in the presence of a protease and an amylase in Small & Mighty liquid detergent. FIG. 6B shows the cleaning performance of various forms of Bsp Man4 in the presence of a protease and an amylase in OMO Color powder detergent.

[0018] FIG. 7A-C provides an alignment of the amino acid sequence of the mature form of Bsp Man4 (SEQ ID NO:8) with the sequences of other microbial mannanases (SEQ ID NOs:15-24). Table 7-1 lists the homologous mannanases by NCBI and SEQ ID NO.

[0019] FIG. 8 provides a phylogenetic tree for Bsp Man4.

[0020] FIG. 9 shows the predicted functional domains of Bsp Man4. The catalytic domain of Bsp Man4 (SEQ ID NO:9) corresponds to residues 11-306 of SEQ ID NO:8. The two predicted catalytic glutamic acid (E) residues are marked. Also shown are the two predicted carbohydrate-binding modules of Bsp Man4.

[0021] FIG. 10 provides the diagrams of protein domains for Bsp Man4 and Bsp Man4 C-terminal truncations.

[0022] FIG. 11A-D provides plasmid maps of pLL007 (aprE-Bsp Man4 1-350), pLL008 (aprE-Bsp Man4 1-475), pLL009 (aprE-Bsp Man4 1-675), and pLL010 (aprE-Bsp Man4 1-850).

[0023] FIG. 12 shows the pH profile of Bsp Man4v2

[0024] FIG. 13 shows the temperature profile of Bsp Man4v2

[0025] FIG. 14 shows the thermostability of Bsp Man4 and Bsp Man4v2

DETAILED DESCRIPTION

I. Introduction

[0026] Described are compositions and methods relating to endo-.beta.-mannanase4 cloned from Bacillus sp SWT81 (Bsp Man4). The compositions and methods are based, in part, on the observation that recombinant Bsp Man4 has glycosyl hydrolase activity in the presence of detergent compositions. This feature of Bsp Man4 makes it well suited for use in a variety of cleaning applications, where the enzyme can hydrolyze mannans in the presence of surfactants and other components found in detergent compositions.

II. Definitions

[0027] Prior to describing the present compositions and methods in detail, the following terms are defined for clarity. Terms and abbreviations not defined should be accorded their ordinary meaning as used in the art:

[0028] As used herein, a "mannan endo-1,4-.beta.-mannosidase," "endo-1,4-.beta.-mannanase," "endo-.beta.-1,4-mannase," ".beta.-mannanase B," ".beta.-1,4-mannan 4-mannanohydrolase," "endo-.beta.-mannanase," ".beta.-D-mannanase," "1,4-.beta.-D-mannan mannanohydrolase," or "endo-.beta.-mannanase" (EC 3.2.1.78) refers to an enzyme capable of the hydrolysis of 1,4-.beta.-D-mannosidic linkages in mannans, galactomannans and glucomannans. Endo-1,4-.beta.-mannanases are members of several families of glycosyl hydrolases, including GH26 and GH5. In particular, endo-.beta.-mannanases constitute a group of polysaccharases that degrade mannans and denote enzymes that are capable of cleaving polyose chains containing mannose units (i.e., are capable of cleaving glycosidic bonds in mannans, glucomannans, galactomannans and galactogluco-mannans). The "endo-.beta.-mannanases" of the present disclosure may possess additional enzymatic activities (e.g., endo-1,4-.beta.-glucanase, 1,4-(3-mannosidase, cellodextrinase activities, etc.).

[0029] As used herein, a "mannanase," "mannosidic enzyme," "mannolytic enzyme," "mannanase enzyme," "mannanase polypeptides," or "mannanase proteins" refers to an enzyme, polypeptide, or protein exhibiting a mannan degrading capability. The mannanase enzyme may be, for example, an endo-.beta.-mannanase, an exo-.beta.-mannanase, or a glycosyl hydrolase. As used herein, mannanase activity may be determined according to any procedure known in the art (See, e.g., Lever, Anal. Biochem, 47:248, 1972; U.S. Pat. No. 6,602,842; and International Publication No. WO 95/35362A1).

[0030] As used herein, "mannans" are polysaccharides having a backbone composed of .beta.-1,4-linked mannose; "glucomannans" are polysaccharides having a backbone of more or less regularly alternating .beta.-1,4 linked mannose and glucose; "galactomannans" and "galactoglucomannans" are mannans and glucomannans with alpha-1,6 linked galactose sidebranches. These compounds may be acetylated. The degradation of galactomannans and galactoglucomannans is facilitated by full or partial removal of the galactose sidebranches. Further the degradation of the acetylated mannans, glucomannans, galactomannans and galactoglucomannans is facilitated by full or partial deacetylation. Acetyl groups can be removed by alkali or by mannan acetylesterases. The oligomers that are released from the mannanases or by a combination of mannanases and alpha-galactosidase and/or mannan acetyl esterases can be further degraded to release free maltose by .beta.-mannosidase and/or .beta.-glucosidase

[0031] As used herein, "catalytic activity" or "activity" describes quantitatively the conversion of a given substrate under defined reaction conditions. The term "residual activity" is defined as the ratio of the catalytic activity of the enzyme under a certain set of conditions to the catalytic activity under a different set of conditions. The term "specific activity" describes quantitatively the catalytic activity per amount of enzyme under defined reaction conditions.

[0032] As used herein, "pH-stability" describes the property of a protein to withstand a limited exposure to pH-values significantly deviating from the pH where its stability is optimal (e.g., more than one pH-unit above or below the pH-optimum, without losing its activity under conditions where its activity is measurable).

[0033] As used herein, the phrase "detergent stability" refers to the stability of a specified detergent composition component (such as a hydrolytic enzyme) in a detergent composition mixture.

[0034] As used herein, a "perhydrolase" is an enzyme capable of catalyzing a reaction that results in the formation of a peracid suitable for applications such as cleaning, bleaching, and disinfecting.

[0035] As used herein, the term "aqueous," as used in the phrases "aqueous composition" and "aqueous environment," refers to a composition that is made up of at least 50% water. An aqueous composition may contain at least 50% water, at least 60% water, at least 70% water, at least 80% water, at least 90% water, at least 95% water, at least 97% water, at least 99% water, or even at least 99% water.

[0036] As used herein, the term "surfactant" refers to any compound generally recognized in the art as having surface active qualities. Surfactants generally include anionic, cationic, nonionic, and zwitterionic compounds, which are further described, herein.

[0037] As used herein, "surface property" is used in reference to electrostatic charge, as well as properties such as the hydrophobicity and hydrophilicity exhibited by the surface of a protein.

[0038] The term "oxidation stability" refers to endo-.beta.-mannanases of the present disclosure that retain a specified amount of enzymatic activity over a given period of time under conditions prevailing during the mannosidic, hydrolyzing, cleaning, or other process disclosed herein, for example while exposed to or contacted with bleaching agents or oxidizing agents. In some embodiments, the endo-.beta.-mannanases retain at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% endo-.beta.-mannanase activity after contact with a bleaching or oxidizing agent over a given time period, for example, at least about 1 minute, about 3 minutes, about 5 minutes, about 8 minutes, about 12 minutes, about 16 minutes, about 20 minutes, etc.

[0039] The term "chelator stability" refers to endo-.beta.-mannanases of the present disclosure that retain a specified amount of enzymatic activity over a given period of time under conditions prevailing during the mannosidic, hydrolyzing, cleaning, or other process disclosed herein, for example while exposed to or contacted with chelating agents. In some embodiments, the endo-.beta.-mannanases retain at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% endo-.beta.-mannanase activity after contact with a chelating agent over a given time period, for example, at least about 10 minutes, about 20 minutes, about 40 minutes, about 60 minutes, about 100 minutes, etc.

[0040] The terms "thermal stability" and "thermostable" refer to endo-.beta.-mannanases of the present disclosure that retain a specified amount of enzymatic activity after exposure to identified temperatures over a given period of time under conditions prevailing during the mannosidic, hydrolyzing, cleaning, or other process disclosed herein, for example, while exposed to altered temperatures. Altered temperatures include increased or decreased temperatures. In some embodiments, the endo-.beta.-mannanases retain at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% endo-.beta.-mannanase activity after exposure to altered temperatures over a given time period, for example, at least about 60 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, etc.

[0041] The term "cleaning activity" refers to the cleaning performance achieved by the endo-.beta.-mannanase under conditions prevailing during the mannosidic, hydrolyzing, cleaning, or other process disclosed herein. In some embodiments, cleaning performance is determined by the application of various cleaning assays concerning enzyme sensitive stains, for example ice cream, ketchup, BBQ sauce, mayonnaise, chocolate milk, body lotion, locust bean gum, or guar gum as determined by various chromatographic, spectrophotometric or other quantitative methodologies after subjection of the stains to standard wash conditions. Exemplary assays include, but are not limited to those described in WO 99/34011, U.S. Pat. No. 6,605,458, and U.S. Pat. No. 6,566,114 (all of which are herein incorporated by reference), as well as those methods included in the Examples.

[0042] As used herein, the terms "clean surface" and "clean textile" refer to a surface or textile respectively that has a percent stain removal of at least 10%, preferably at least 15%, 20%, 25%, 30%, 35%, or 40% of a soiled surface or textile.

[0043] The term "cleaning effective amount" of an endo-.beta.-mannanase refers to the quantity of endo-.beta.-mannanase described hereinbefore that achieves a desired level of enzymatic activity in a specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular endo-.beta.-mannanase used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, etc.

[0044] The term "cleaning adjunct materials," as used herein, means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, granule, powder, bar, paste, spray, tablet, gel, or foam composition), which materials are also preferably compatible with the endo-.beta.-mannanase enzyme used in the composition. In some embodiments, granular compositions are in "compact" form, while in other embodiments, the liquid compositions are in a "concentrated" form.

[0045] As used herein, "cleaning compositions" and "cleaning formulations" refer to admixtures of chemical ingredients that find use in the removal of undesired compounds (e.g., soil or stains) from items to be cleaned, such as fabric, dishes, contact lenses, other solid surfaces, hair, skin, teeth, and the like. The composition or formulations may be in the form of a liquid, gel, granule, powder, or spray, depending on the surface, item or fabric to be cleaned, and the desired form of the composition or formulation.

[0046] As used herein, the terms "detergent composition" and "detergent formulation" refer to mixtures of chemical ingredients intended for use in a wash medium for the cleaning of soiled objects. Detergent compositions/formulations generally include at least one surfactant, and may optionally include hydrolytic enzymes, oxido-reductases, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and solubilizers.

[0047] As used herein, "laundry composition" or "laundry detergent" refers to all forms of compositions for cleaning textiles, including but not limited to granular and liquid forms. In some embodiments, the laundry composition is a composition that finds use in an electric clothes washer. It is not intended that the present disclosure be limited to any particular type or laundry composition. Indeed, the present disclosure finds use in cleaning many fabrics.

[0048] As used herein, "dishwashing composition" refers to all forms of compositions for cleaning dishware, including cutlery, including but not limited to granular and liquid forms. In some embodiments, the dishwashing composition is an "automatic dishwashing" composition that finds use in automatic dish washing machines. It is not intended that the present disclosure be limited to any particular type or dishware composition. Indeed, the present disclosure finds use in cleaning dishware (e.g., dishes including, but not limited to plates, cups, glasses, bowls, etc.) and cutlery (e.g., utensils including, but not limited to spoons, knives, forks, serving utensils, etc.) of any material, including but not limited to ceramics, plastics, metals, china, glass, acrylics, etc. The term "dishware" is used herein in reference to both dishes and cutlery.

[0049] As used herein, the term "bleaching" refers to the treatment of a material (e.g., fabric, laundry, pulp, etc.) or surface for a sufficient length of time and under appropriate pH and temperature conditions to effect a brightening (i.e., whitening) and/or cleaning of the material. Examples of chemicals suitable for bleaching include but are not limited to ClO.sub.2, H.sub.2O.sub.2, peracids, NO.sub.2, etc.

[0050] As used herein, "wash performance" of a variant endo-.beta.-mannanase refers to the contribution of a variant endo-.beta.-mannanase to washing that provides additional cleaning performance to the detergent without the addition of the variant endo-.beta.-mannanase to the composition. Wash performance is compared under relevant washing conditions.

[0051] The term "relevant washing conditions" is used herein to indicate the conditions, particularly washing temperature, time, washing mechanics, sud concentration, type of detergent, and water hardness, actually used in households in a dish or laundry detergent market segment.

[0052] As used herein, the term "disinfecting" refers to the removal of contaminants from the surfaces, as well as the inhibition or killing of microbes on the surfaces of items. It is not intended that the present disclosure be limited to any particular surface, item, or contaminant(s) or microbes to be removed.

[0053] The "compact" form of the cleaning compositions herein is best reflected by density and, in terms of composition, by the amount of inorganic filler salt. Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, the filler salts are present in substantial amounts, typically about 17 to about 35% by weight of the total composition. In contrast, in compact compositions, the filler salt is present in amounts not exceeding about 15% of the total composition. In some embodiments, the filler salt is present in amounts that do not exceed about 10%, or more preferably, about 5%, by weight of the composition. In some embodiments, the inorganic filler salts are selected from the alkali and alkaline-earth-metal salts of sulfates and chlorides. In some embodiments, a preferred filler salt is sodium sulfate.

[0054] As used herein, the terms "textile" or "textile material" refer to woven fabrics, as well as staple fibers and filaments suitable for conversion to or use as yarns, woven, knit, and non-woven fabrics. The term encompasses yarns made from natural, as well as synthetic (e.g., manufactured) fibers.

[0055] As used herein, the terms "purified" and "isolated" refer to the physical separation of a subject molecule, such as Bsp Man4, from its native source (e.g., Bacillus sp.) or other molecules, such as proteins, nucleic acids, lipids, media components, and the like. Once purified or isolated, a subject molecule may represent at least 50%, and even at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or more, of the total amount of material in a sample (wt/wt).

[0056] As used herein, a "polypeptide" refers to a molecule comprising a plurality of amino acids linked through peptide bonds. The terms "polypeptide," "peptide," and "protein" are used interchangeably. Proteins maybe optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, sulfonated, pegylated, and the like) to add functionality. Where such amino acid sequences exhibit activity, they may be referred to as an "enzyme." The conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N.fwdarw.C).

[0057] The terms "polynucleotide" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single-stranded or double-stranded, and may have chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences which encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5'-to-3' orientation.

[0058] As used herein, the terms "wild-type" and "native" refer to polypeptides or polynucleotides that are found in nature.

[0059] The terms, "wild-type," "parental," or "reference," with respect to a polypeptide, refer to a naturally-occurring polypeptide that does not include a man-made substitution, insertion, or deletion at one or more amino acid positions. Similarly, the terms "wild-type," "parental," or "reference," with respect to a polynucleotide, refer to a naturally-occurring polynucleotide that does not include a man-made nucleoside change. However, note that a polynucleotide encoding a wild-type, parental, or reference polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wild-type, parental, or reference polypeptide.

[0060] As used herein, a "variant polypeptide" refers to a polypeptide that is derived from a parent (or reference) polypeptide by the substitution, addition, or deletion, of one or more amino acids, typically by recombinant DNA techniques. Variant polypeptides may differ from a parent polypeptide by a small number of amino acid residues and may be defined by their level of primary amino acid sequence homology/identity with a parent polypeptide. Preferably, variant polypeptides have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity with a parent polypeptide.

[0061] Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See, e.g., Altschul et al. [1990] J. Mol. Biol. 215:403-410; Henikoff et al. [1989] Proc. Natl. Acad. Sci. USA 89:10915; Karin et al. [1993] Proc. Natl. Acad. Sci. USA 90:5873; and Higgins et al. [1988] Gene 73:237-244). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Databases may also be searched using FASTA (Pearson et al. [1988] Proc. Natl. Acad. Sci. USA 85:2444-2448). One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.

[0062] As used herein, a "variant polynucleotide" encodes a variant polypeptide, has a specified degree of homology/identity with a parent polynucleotide, or hybridized under stringent conditions to a parent polynucleotide or the complement, thereof. Preferably, a variant polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% nucleotide sequence identity with a parent polynucleotide. Methods for determining percent identity are known in the art and described immediately above.

[0063] The term "derived from" encompasses the terms "originated from," "obtained from," "obtainable from," "isolated from," and "created from," and generally indicates that one specified material find its origin in another specified material or has features that can be described with reference to the another specified material.

[0064] As used herein, the term "hybridization" refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing, as known in the art.

[0065] As used herein, the phrase "hybridization conditions" refers to the conditions under which hybridization reactions are conducted. These conditions are typically classified by degree of "stringency" of the conditions under which hybridization is measured. The degree of stringency can be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about Tm-5.degree. C. (5.degree. below the Tm of the probe); "high stringency" at about 5-10.degree. below the Tm; "intermediate stringency" at about 10-20.degree. below the Tm of the probe; and "low stringency" at about 20-25.degree. below the Tm. Alternatively, or in addition, hybridization conditions can be based upon the salt or ionic strength conditions of hybridization and/or one or more stringency washes, e.g.,: 6.times.SSC=very low stringency; 3.times.SSC=low to medium stringency; 1.times.SSC=medium stringency; and 0.5.times.SSC=high stringency. Functionally, maximum stringency conditions may be used to identify nucleic acid sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions are used to identify nucleic acid sequences having about 80% or more sequence identity with the probe. For applications requiring high selectivity, it is typically desirable to use relatively stringent conditions to form the hybrids (e.g., relatively low salt and/or high temperature conditions are used). As used herein, stringent conditions are defined as 50.degree. C. and 0.2.times.SSC (1.times.SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.0).

[0066] The phrases "substantially similar" and "substantially identical" in the context of at least two nucleic acids or polypeptides means that a polynucleotide or polypeptide comprises a sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or even at least about 99% identical to a parent or reference sequence, or does not include amino acid substitutions, insertions, deletions, or modifications made only to circumvent the present description without adding functionality.

[0067] As used herein, an "expression vector" refers to a DNA construct containing a DNA sequence that encodes a specified polypeptide and is operably linked to a suitable control sequence capable of effecting the expression of the polypeptides in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.

[0068] The term "recombinant," refers to genetic material (i.e., nucleic acids, the polypeptides they encode, and vectors and cells comprising such polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that of another gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at a decreased or elevated levels, expressing a gene conditionally or constitutively in manner different from its natural expression profile, and the like. Generally recombinant nucleic acids, polypeptides, and cells based thereon, have been manipulated by man such that they are not identical to related nucleic acids, polypeptides, and cells found in nature.

[0069] A "signal sequence" refers to a sequence of amino acids bound to the N-terminal portion of a polypeptide, and which facilitates the secretion of the mature form of the protein from the cell. The mature form of the extracellular protein lacks the signal sequence which is cleaved off during the secretion process.

[0070] The term "selective marker" or "selectable marker" refers to a gene capable of expression in a host cell that allows for ease of selection of those hosts containing an introduced nucleic acid or vector. Examples of selectable markers include but are not limited to antimicrobial substances (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage, on the host cell.

[0071] The term "regulatory element" as used herein refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Additional regulatory elements include splicing signals, polyadenylation signals and termination signals.

[0072] As used herein, "host cells" are generally prokaryotic or eukaryotic hosts which are transformed or transfected with vectors constructed using recombinant DNA techniques known in the art. Transformed host cells are capable of either replicating vectors encoding the protein variants or expressing the desired protein variant. In the case of vectors which encode the pre- or pro-form of the protein variant, such variants, when expressed, are typically secreted from the host cell into the host cell medium.

[0073] The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means transformation, transduction or transfection. Means of transformation include protoplast transformation, calcium chloride precipitation, electroporation, naked DNA, and the like as known in the art. (See, Chang and Cohen [1979] Mol. Gen. Genet. 168:111-115; Smith et al. [1986] Appl. Env. Microbiol. 51:634; and the review article by Ferrari et al., in Harwood, Bacillus, Plenum Publishing Corporation, pp. 57-72, 1989).

[0074] The terms "selectable marker" or "selectable gene product" as used herein refer to the use of a gene, which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed.

[0075] Other technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains (See, e.g., Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY 1994; and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY 1991).

[0076] The singular terms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise.

[0077] As used herein in connection with a numerical value, the term "about" refers to a range of -10% to +10% of the numerical value. For instance, the phrase a "pH value of about 6" refers to pH values of from 5.4 to 6.6.

[0078] Headings are provided for convenience and should not be construed as limitations. The description included under one heading may apply to the specification as a whole.

III. Bsp Man4 Polypeptides, Polynucleotides, Vectors, and Host Cells

[0079] A. Bsp Man4 Polypeptides

[0080] In one aspect, the present compositions and methods provide a recombinant Bsp Man4 endo-.beta.-mannanase polypeptide, fragments thereof, or variants thereof. An exemplary Bsp Man4 polypeptide was recombinantly expressed from a polynucleotide obtained from Bacillus sp. The mature Bsp Man4 polypeptide has the amino acid sequence set forth as SEQ ID NO:8. Similar, substantially identical Bsp Man4 polypeptides may occur in nature, e.g., in other strains or isolates of Bacillus. These and other Bsp Man4 polypeptides are encompassed by the present compositions and methods. Bsp Man4 polypeptides of the present invention include truncated forms of Bsp Man4, including C-terminal truncations, that retain mannanase activity. Included amongst these polypeptides are the polypeptides as describes in the Examples and shown as SEQ ID NOs:6-14 and 30-49.

[0081] In some embodiments, the isolated Bsp Man4 polypeptide is a variant Bsp Man4 polypeptide having a specified degree of amino acid sequence identity to the exemplified Bsp Man4 polypeptide, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:8. Sequence identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0082] In some embodiments, the isolated Bsp Man4 polypeptide is a variant Bsp Man4 polypeptide having a specified degree of amino acid sequence identity to the exemplified Bsp Man4 polypeptide, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:6-14 or 30-49. Sequence identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0083] In certain embodiments, the Bsp Man4 polypeptides are produced recombinantly, while in others the Bsp Man4 polypeptides are produced synthetically, or are purified from a native source (Bacillus sp.).

[0084] In certain other embodiments, the isolated Bsp Man4 polypeptide includes substitutions that do not substantially affect the structure and/or function of the polypeptide. Exemplary substitutions are conservative mutations, as summarized in Table I.

TABLE-US-00001 TABLE I Amino Acid Substitutions Original Residue Code Acceptable Substitutions Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, beta-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid, D-or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

[0085] Substitutions involving naturally occurring amino acids are generally made by mutating a nucleic acid encoding a recombinant Bsp Man4 polypeptide, and then expressing the variant polypeptide in an organism. Substitutions involving non-naturally occurring amino acids or chemical modifications to amino acids are generally made by chemically modifying a recombinant Bsp Man4 polypeptide after it has been synthesized by an organism.

[0086] In some embodiments, variant isolated Bsp Man4 polypeptides are substantially identical to SEQ ID NO:8, meaning that they do not include amino acid substitutions, insertions, or deletions that do not significantly affect the structure, function, or expression of the polypeptide. Such variant isolated Bsp Man4 polypeptides include those designed only to circumvent the present description.

[0087] In some embodiments, the isolated Bsp Man4 polypeptide (including a variant thereof) has 1,4-.beta.-D-mannosidic hydrolase activity, which includes mannanase, endo-1,4-(3-D-mannanase, exo-1,4-.beta.-D-mannanasegalactomannanase, and/or glucomannanase activity. 1,4-.beta.-D-mannosidic hydrolase activity can be determined and measured using the assays described herein, or by other assays known in the art. In some embodiments, the isolated Bsp Man4 polypeptide has activity in the presence of a detergent composition.

[0088] Bsp Man4 polypeptides include fragments of "full-length" Bsp Man4 polypeptides that retain 1,4-.beta.-D-mannosidic hydrolase activity. Such fragments preferably retain the active site of the full-length polypeptides but may have deletions of non-critical amino acid residues. The activity of fragments can readily be determined using the assays described, herein, or by other assays known in the art. In some embodiments, the fragments of Bsp Man4 polypeptides retain 1,4-.beta.-D-mannosidic hydrolase activity in the presence of a detergent composition. In some embodiments, the Bsp Man4 polypeptides comprise the catalytic domain of Bsp Man4 (SEQ ID NO:9), or a catalytic domain that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:9.

[0089] In some embodiments, the Bsp Man4 amino acid sequences and derivatives are produced as a N- and/or C-terminal fusion protein, for example to aid in extraction, detection and/or purification and/or to add functional properties to the Bsp Man4 polypeptides. Examples of fusion protein partners include, but are not limited to, glutathione-S-transferase (GST), 6.times.His, GAL4 (DNA binding and/or transcriptional activation domains), FLAG, MYC, BCE103 (WO 2010/044786), or other tags well known to anyone skilled in the art. In some embodiments, a proteolytic cleavage site is provided between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably, the fusion protein does not hinder the activity of the isolated Bsp Man4 polypeptide.

[0090] In some embodiments, the isolated Bsp Man4 polypeptide is fused to a functional domain including a leader peptide, propeptide, one or more binding domains (modules) and/or catalytic domain. Suitable binding domains include, but are not limited to, carbohydrate-binding modules (e.g., CBM) of various specificities, providing increased affinity to carbohydrate components present during the application of the isolated Bsp Man4 polypeptide. As described herein, the CBM and catalytic domain of the Bsp Man4 polypeptide are operably linked.

[0091] A carbohydrate-binding module (CBM) is defined as a contiguous amino acid sequence within a carbohydrate-active enzyme with a discreet fold having carbohydrate-binding activity. A few exceptions are CBMs in cellulosomal scaffoldin proteins and rare instances of independent putative CBMs. The requirement of CBMs existing as modules within larger enzymes sets this class of carbohydrate-binding protein apart from other non-catalytic sugar binding proteins such as lectins and sugar transport proteins. CBMs were previously classified as cellulose-binding domains (CBDs) based on the initial discovery of several modules that bound cellulose (Tomme et al., Eur J Biochem, 170:575-581, 1988; and Gilkes et al., J Biol Chem, 263:10401-10407, 1988). However, additional modules in carbohydrate-active enzymes are continually being found that bind carbohydrates other than cellulose yet otherwise meet the CBM criteria, hence the need to reclassify these polypeptides using more inclusive terminology. Previous classification of cellulose-binding domains was based on amino acid similarity. Groupings of CBDs were called "Types" and numbered with roman numerals (e.g. Type I or Type II CBDs). In keeping with the glycoside hydrolase classification, these groupings are now called families and numbered with Arabic numerals. Families 1 to 13 are the same as Types Ito XIII (Tomme et al., in Enzymatic Degradation of Insoluble Polysaccharides (Saddler, J. N. & Penner, M., eds.), Cellulose-binding domains: classification and properties. pp. 142-163, American Chemical Society, Washington, 1995). A detailed review on the structure and binding modes of CBMs can be found in (Boraston et al., Biochem J, 382:769-81, 2004). The family classification of CBMs is expected to: aid in the identification of CBMs, in some cases, predict binding specificity, aid in identifying functional residues, reveal evolutionary relationships and possibly be predictive of polypeptide folds. Because the fold of proteins is better conserved than their sequences, some of the CBM families can be grouped into superfamilies or clans. The current CBM families are 1-63. CBMs/CBDs have also been found in algae, e.g., the red alga Porphyra purpurea as a non-hydrolytic polysaccharide-binding protein. However, most of the CBDs are from cellullases and xylanases. CBDs are found at the N- and C-termini of proteins or are internal. Enzyme hybrids are known in the art (See e.g., WO 90/00609 and WO 95/16782) and may be prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the cellulose-binding domain ligated, with or without a linker, to a DNA sequence encoding a disclosed Bsp Man4 polypeptide and growing the host cell to express the fused gene. Enzyme hybrids may be described by the following formula:

CBM-MR-X or X-MR-CBM

[0092] In the above formula, the CBM is the N-terminal or the C-terminal region of an amino acid sequence corresponding to at least the carbohydrate-binding module; MR is the middle region (the linker), and may be a bond, or a short linking group preferably of from about 2 to about 100 carbon atoms, more preferably of from 2 to 40 carbon atoms; or is preferably from about 2 to about 100 amino acids, more preferably from 2 to 40 amino acids; and X is an N-terminal or C-terminal region of a disclosed Bsp Man4 polypeptide having mannanase catalytic activity. In addition, a mannanase may contain more than one CBM or other module(s)/domain(s) of non-glycolytic function. The terms "module" and "domain" are used interchangeably in the present disclosure.

[0093] Suitable enzymatically active domains possess an activity that supports the action of the isolated Bsp Man4 polypeptide in producing the desired product. Non-limiting examples of catalytic domains include: cellulases, hemicellulases such as xylanase, exo-mannanases, glucanases, arabinases, galactosidases, pectinases, and/or other activities such as proteases, lipases, acid phosphatases and/or others or functional fragments thereof. Fusion proteins are optionally linked to the isolated Bsp Man4 polypeptide through a linker sequence that simply joins the Bsp Man4 polypeptide and the fusion domain without significantly affecting the properties of either component, or the linker optionally has a functional importance for the intended application.

[0094] Alternatively, the isolated Bsp Man4 polypeptides described herein are used in conjunction with one or more additional proteins of interest. Non-limiting examples of proteins of interest include: hemicellulases, exo-.beta.-mannanases, alpha-galactosidases, beta-galactosidases, lactases, beta-glucanases, endo-beta-1,4-glucanases, cellulases, xylosidases, xylanases, xyloglucanases, xylan acetyl-esterases, galactanases, exo-mannanases, pectinases, pectin lyases, pectinesterases, polygalacturonases, arabinases, rhamnogalacturonases, laccases, reductases, oxidases, phenoloxidases, ligninases, proteases, amylases, phosphatases, lipolytic enzymes, cutinases and/or other enzymes.

[0095] In other embodiments, the isolated Bsp Man4 polypeptide is fused to a signal peptide for directing the extracellular secretion of the isolated Bsp Man4 polypeptide. For example, in certain embodiments, the signal peptide is the native Bsp Man4 signal peptide. In other embodiments, the signal peptide is a non-native signal peptide such as the B. subtilis AprE signal peptide. In some embodiments, the isolated Bsp Man4 polypeptide has an N-terminal extension of Ala-Gly-Lys between the mature form and the signal peptide.

[0096] In some embodiments, the isolated Bsp Man4 polypeptide is expressed in a heterologous organism, i.e., an organism other than Bacillus agaradhaerens. Exemplary heterologous organisms are Gram(+) bacteria such as Bacillus subtilis, Bacillus lichenifonnis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans, or Streptomyces murinus; Gram(-) bacteria such as Escherichia coli.; yeast such as Saccharomyces spp. or Schizosaccharomyces spp., e.g. Saccharomyces cerevisiae; and filamentous fungi such as Aspergillus spp., e.g., Aspergillus oryzae or Aspergillus niger, and Trichoderma reesei. Methods from transforming nucleic acids into these organisms are well known in the art. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.

[0097] In particular embodiments, the isolated Bsp Man4 polypeptide is expressed in a heterologous organism as a secreted polypeptide, in which case, the compositions and method encompass a method for expressing a Bsp Man4 polypeptide as a secreted polypeptide in a heterologous organism.

[0098] B. Bsp Man4 Polynucleotides

[0099] Another aspect of the compositions and methods is a polynucleotide that encodes an isolated Bsp Man4 polypeptide (including variants and fragments, thereof), provided in the context of an expression vector for directing the expression of a Bsp Man4 polypeptide in a heterologous organism, such as those identified, herein. The polynucleotide that encodes a Bsp Man4 polypeptide may be operably-linked to regulatory elements (e.g., a promoter, terminator, enhancer, and the like) to assist in expressing the encoded polypeptides.

[0100] An exemplary polynucleotide sequence encoding a Bsp Man4 polypeptide has the nucleotide sequence of SEQ ID NO:1. Similar, including substantially identical, polynucleotides encoding Bsp Man4 polypeptides and variants may occur in nature, e.g., in other strains or isolates of Bacillus. In view of the degeneracy of the genetic code, it will be appreciated that polynucleotides having different nucleotide sequences may encode the same Bsp Man4 polypeptides, variants, or fragments.

[0101] In some embodiments, polynucleotides encoding Bsp Man4 polypeptides have a specified degree of amino acid sequence identity to the exemplified polynucleotide encoding a Bsp Man4 polypeptide, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the polynucleotides encode Bsp Man4 polypeptides comprising the catalytic domain of Bsp Man4 (SEQ ID NO:9), or a catalytic domain that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:9. Homology can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0102] In some embodiments, the polynucleotide that encodes a Bsp Man4 polypeptide is fused in frame behind (i.e., downstream of) a coding sequence for a signal peptide for directing the extracellular secretion of a Bsp Man4 polypeptide. Heterologous signal sequences include those from bacterial cellulase genes. Expression vectors may be provided in a heterologous host cell suitable for expressing a Bsp Man4 polypeptide, or suitable for propagating the expression vector prior to introducing it into a suitable host cell.

[0103] In some embodiments, polynucleotides encoding Bsp Man4 polypeptides hybridize to the exemplary polynucleotide of SEQ ID NO:1 (or the complement thereof) under specified hybridization conditions. Exemplary conditions are stringent condition and highly stringent conditions, which are described, herein.

[0104] Bsp Man4 polynucleotides may be naturally occurring or synthetic (i.e., man-made), and may be codon-optimized for expression in a different host, mutated to introduce cloning sites, or otherwise altered to add functionality.

[0105] C. Bsp Man4 Vectors and Host Cells

[0106] In order to produce a disclosed Bsp Man4 polypeptide, the DNA encoding the polypeptide can be chemically synthesized from published sequences or obtained directly from host cells harboring the gene (e.g., by cDNA library screening or PCR amplification). In some embodiments, the Bsp Man4 polynucleotide is included in an expression cassette and/or cloned into a suitable expression vector by standard molecular cloning techniques. Such expression cassettes or vectors contain sequences that assist initiation and termination of transcription (e.g., promoters and terminators), and generally contain a selectable marker.

[0107] The expression cassette or vector is introduced in a suitable expression host cell, which then expresses the corresponding Bsp Man4 polynucleotide. Particularly suitable expression hosts are bacterial expression host genera including Escherichia (e.g., Escherichia coli), Pseudomonas (e.g., P. fluorescens or P. stutzerei), Proteus (e.g., Proteus mirabilis), Ralstonia (e.g., Ralstonia eutropha), Streptomyces, Staphylococcus (e.g., S. carnosus), Lactococcus (e.g., L. lactis), or Bacillus (subtilis, megaterium, lichenifonnis, etc.). Also particularly suitable are yeast expression hosts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis or Pichia pastoris. Especially suited are fungal expression hosts such as Aspergillus niger, Chrysosporium lucknowense, Aspergillus (e.g., A. oryzae, A. niger, A. nidulans, etc.) or Trichoderma reesei. Also suited are mammalian expression hosts such as mouse (e.g., NS0), Chinese Hamster Ovary (CHO) or Baby Hamster Kidney (BHK) cell lines. Other eukaryotic hosts such as insect cells or viral expression systems (e.g., bacteriophages such as M13, T7 phage or Lambda, or viruses such as Baculovirus) are also suitable for producing the Bsp Man4 polypeptide.

[0108] Promoters and/or signal sequences associated with secreted proteins in a particular host of interest are candidates for use in the heterologous production and secretion of endo-.beta.-mannanases in that host or in other hosts. As an example, in filamentous fungal systems, the promoters that drive the genes for cellobiohydrolase I (cbh1), glucoamylase A (glaA), TAKA-amylase (amyA), xylanase (exlA), the gpd-promoter cbh1, cbhll, endoglucanase genes EGI-EGV, Cel61B, Cel74A, eg11-eg15, gpd promoter, Pgk1, pki1, EF-1alpha, tef1, cDNA1 and hex1 are particularly suitable and can be derived from a number of different organisms (e.g., A. niger, T. reesei, A. oryzae, A. awamori and A. nidulans). In some embodiments, the Bsp Man4 polynucleotide is recombinantly associated with a polynucleotide encoding a suitable homologous or heterologous signal sequence that leads to secretion of the Bsp Man4 polypeptide into the extracellular (or periplasmic) space, thereby allowing direct detection of enzyme activity in the cell supernatant (or periplasmic space or lysate). Particularly suitable signal sequences for Escherichia coli, other Gram negative bacteria and other organisms known in the art include those that drive expression of the HlyA, DsbA, Pbp, PhoA, Pe1B, OmpA, OmpT or M13 phage Gill genes. For Bacillus subtilis, Gram-positive organisms and other organisms known in the art, particularly suitable signal sequences further include those that drive expression of the AprE, NprB, Mpr, AmyA, AmyE, Blac, SacB, and for S. cerevisiae or other yeast, include the killer toxin, Bar1, Suc2, Mating factor alpha, Inu1A or Ggp1p signal sequence. Signal sequences can be cleaved by a number of signal peptidases, thus removing them from the rest of the expressed protein. In some embodiments, the rest of the Bsp Man4 polypeptide is expressed alone or as a fusion with other peptides, tags or proteins located at the N- or C-terminus (e.g., 6.times.His, HA or FLAG tags). Suitable fusions include tags, peptides or proteins that facilitate affinity purification or detection (e.g., BCE103, 6.times.His, HA, chitin binding protein, thioredoxin or FLAG tags), as well as those that facilitate expression, secretion or processing of the target endo-.beta.-mannanase. Suitable processing sites include enterokinase, STE13, Kex2 or other protease cleavage sites for cleavage in vivo or in vitro.

[0109] Bsp Man4 polynucleotides are introduced into expression host cells by a number of transformation methods including, but not limited to, electroporation, lipid-assisted transformation or transfection ("lipofection"), chemically mediated transfection (e.g., CaCl and/or CaP), lithium acetate-mediated transformation (e.g., of host-cell protoplasts), biolistic "gene gun" transformation, PEG-mediated transformation (e.g., of host-cell protoplasts), protoplast fusion (e.g., using bacterial or eukaryotic protoplasts), liposome-mediated transformation, Agrobacterium tumefaciens, adenovirus or other viral or phage transformation or transduction.

[0110] Alternatively, the Bsp Man4 polypeptides are expressed intracellularly. Optionally, after intracellular expression of the enzyme variants, or secretion into the periplasmic space using signal sequences such as those mentioned above, a permeabilisation or lysis step can be used to release the Bsp Man4 polypeptide into the supernatant. The disruption of the membrane barrier is effected by the use of mechanical means such as ultrasonic waves, pressure treatment (French press), cavitation or the use of membrane-digesting enzymes such as lysozyme or enzyme mixtures. As a further alternative, the polynucleotides encoding the Bsp Man4 polypeptide are expressed by use of a suitable cell-free expression system. In cell-free systems, the polynucleotide of interest is typically transcribed with the assistance of a promoter, but ligation to form a circular expression vector is optional. In other embodiments, RNA is exogenously added or generated without transcription and translated in cell free systems.

IV. Activities of Bsp Man4

[0111] The isolated Bsp Man4 polypeptides disclosed herein may have enzymatic activity over a broad range of pH conditions. In certain embodiments the disclosed Bsp Man4 polypeptides have enzymatic activity from about pH 4.0 to about pH 11.5. In preferred embodiments, the Bsp Man4 polypeptides have substantial enzymatic activity from about pH 6.0 to about pH 8.5. It should be noted that the pH values described herein may vary by .+-.0.2. For example a pH value of 8.0 could vary from pH 7.8 to pH 8.2.

[0112] The isolated Bsp Man4 polypeptides disclosed herein may have enzymatic activity over a wide range of temperatures, e.g., from 35.degree. C. or lower to about 75.degree. C. In certain embodiments, the Bsp Man4 polypeptides have substantial enzymatic activity at a temperature range of about 55.degree. C. to about 65.degree. C. It should be noted that the temperature values described herein may vary by .+-.0.2.degree. C. For example a temperature of 50.degree. C. could vary from 49.8.degree. C. to 50.2.degree. C.

[0113] As shown in Example 5, the Bsp Man4 polypeptide had cleaning performance against locust bean gum and guar gum in the presence of proteases. Moreover, Bsp Man4 showed hydrolysis activity against exemplary gum stained material, in the presence of both powder and liquid detergent. Accordingly, in certain embodiments, any of the isolated Bsp Man4 polypeptides described herein may hydrolyze mannan substrates that include, but are not limited to, locust bean gum, guar gum, and combinations thereof.

V. Detergent Compositions Comprising a Bsp Man4 Polypeptide

[0114] An aspect of the compositions and methods disclosed herein is a detergent composition comprising an isolated Bsp Man4 polypeptide (including variants or fragments, thereof) and methods for using such compositions in cleaning applications. Cleaning applications include, but are not limited to, laundry or textile cleaning, laundry or textile softening, dishwashing (manual and automatic), stain pre-treatment, and the like. Particular applications are those where mannans (e.g., locust bean gum, guar gum, etc.) are a component of the soils or stains to be removed. Detergent compositions typically include an effective amount of any of the Bsp Man4 polypeptides described herein, e.g., at least 0.0001 weight percent, from about 0.0001 to about 1, from about 0.001 to about 0.5, from about 0.01 to about 0.1 weight percent, or even from about 0.1 to about 1 weight percent, or more. An effective amount of a Bsp Man4 polypeptide in the detergent composition results in the Bsp Man4 polypeptide having enzymatic activity sufficient to hydrolyze a mannan-containing substrate, such as locust bean gum, guar gum, or combinations thereof.

[0115] Additionally, detergent compositions having a concentration from about 0.4 g/L to about 2.2 g/L, from about 0.4 g/L to about 2.0 g/L, from about 0.4 g/L to about 1.7 g/L, from about 0.4 g/L to about 1.5 g/L, from about 0.4 g/L to about 1 g/L, from about 0.4 g/L to about 0.8 g/L, or from about 0.4 g/L to about 0.5 g/L may be mixed with an effective amount of an isolated Bsp Man4 polypeptide. The detergent composition may also be present at a concentration of about 0.4 ml/L to about 2.6 ml/L, from about 0.4 ml/L to about 2.0 ml/L, from about 0.4 ml/L to about 1.5 m/L, from about 0.4 ml/L to about 1 ml/L, from about 0.4 ml/L to about 0.8 ml/L, or from about 0.4 ml/L to about 0.5 ml/L.

[0116] Unless otherwise noted, all component or composition levels provided herein are made in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources. Enzyme components weights are based on total active protein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated. In the exemplified detergent compositions, the enzymes levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the detergent ingredients are expressed by weight of the total compositions.

[0117] In some embodiments, the detergent composition comprises one or more surfactants, which may be non-ionic, semi-polar, anionic, cationic, zwitterionic, or combinations and mixtures thereof. The surfactants are typically present at a level of from about 0.1% to 60% by weight. Exemplary surfactants include but are not limited to sodium dodecylbenzene sulfonate, C12-14 pareth-7, C12-15 pareth-7, sodium C12-15 pareth sulfate, C14-15 pareth-4, sodium laureth sulfate (e.g., Steol CS-370), sodium hydrogenated cocoate, C12 ethoxylates (Alfonic 1012-6, Hetoxol LA7, Hetoxol LA4), sodium alkyl benzene sulfonates (e.g., Nacconol 90G), and combinations and mixtures thereof.

[0118] Anionic surfactants that may be used with the detergent compositions described herein include but are not limited to linear alkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% of nonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide (e.g., as described in WO 92/06154), and combinations and mixtures thereof.

[0119] Nonionic surfactants that may be used with the detergent compositions described herein include but are not limited to polyoxyethylene esters of fatty acids, polyoxyethylene sorbitan esters (e.g., TWEENs), polyoxyethylene alcohols, polyoxyethylene isoalcohols, polyoxyethylene ethers (e.g., TRITONs and BRIJ), polyoxyethylene esters, polyoxyethylene-p-tert-octylphenols or octylphenyl-ethylene oxide condensates (e.g., NONIDET P40), ethylene oxide condensates with fatty alcohols (e.g., LUBROL), polyoxyethylene nonylphenols, polyalkylene glycols (SYNPERONIC F108), sugar-based surfactants (e.g., glycopyranosides, thioglycopyranosides), and combinations and mixtures thereof.

[0120] The detergent compositions disclosed herein may have mixtures that include, but are not limited to 5-15% anionic surfactants, <5% nonionic surfactants, cationic surfactants, phosphonates, soap, enzymes, perfume, butylphenyl methylptopionate, geraniol, zeolite, polycarboxylates, hexyl cinnamal, limonene, cationic surfactants, citronellol, and benzisothiazolinone.

[0121] Detergent compositions may additionally include one or more detergent builders or builder systems, a complexing agent, a polymer, a bleaching system, a stabilizer, a foam booster, a suds suppressor, an anti-corrosion agent, a soil-suspending agent, an anti-soil redeposition agent, a dye, a bactericide, a hydrotope, a tarnish inhibitor, an optical brightener, a fabric conditioner, and a perfume. The detergent compositions may also include enzymes, including but not limited to proteases, amylases, cellulases, lipases, pectin degrading enzymes, xyloglucanases, or additional carboxylic ester hydrolases. The pH of the detergent compositions should be neutral to basic, as described herein.

[0122] In some embodiments incorporating at least one builder, the detergent compositions comprise at least about 1%, from about 3% to about 60% or even from about 5% to about 40% builder by weight of the cleaning composition. Builders may include, but are not limited to, the alkali metals, ammonium and alkanolammonium salts of polyphosphates, alkali metal silicates, alkaline earth and alkali metal carbonates, aluminosilicates, polycarboxylate compounds, ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1, 3, 5-trihydroxy benzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, the various alkali metals, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof. Indeed, it is contemplated that any suitable builder will find use in various embodiments of the present disclosure.

[0123] In some embodiments, the builders form water-soluble hardness ion complexes (e.g., sequestering builders), such as citrates and polyphosphates (e.g., sodium tripolyphosphate and sodium tripolyphospate hexahydrate, potassium tripolyphosphate, and mixed sodium and potassium tripolyphosphate, etc.). It is contemplated that any suitable builder will find use in the present disclosure, including those known in the art (See, e.g., EP 2 100 949).

[0124] As indicated herein, in some embodiments, the cleaning compositions described herein further comprise adjunct materials including, but not limited to surfactants, builders, bleaches, bleach activators, bleach catalysts, other enzymes, enzyme stabilizing systems, chelants, optical brighteners, soil release polymers, dye transfer agents, dispersants, suds suppressors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescers, fabric conditioners, hydrolyzable surfactants, preservatives, anti-oxidants, anti-shrinkage agents, anti-wrinkle agents, germicides, fungicides, color speckles, silvercare, anti-tarnish and/or anti-corrosion agents, alkalinity sources, solubilizing agents, carriers, processing aids, pigments, and pH control agents (See, e.g., U.S. Pat. Nos. 6,610,642; 6,605,458; 5,705,464; 5,710,115; 5,698,504; 5,695,679; 5,686,014; and 5,646,101; all of which are incorporated herein by reference). Embodiments of specific cleaning composition materials are exemplified in detail below. In embodiments in which the cleaning adjunct materials are not compatible with the Bsp Man4 variants in the cleaning compositions, suitable methods of keeping the cleaning adjunct materials and the endo-.beta.-mannanase(s) separated (i.e., not in contact with each other), until combination of the two components is appropriate, are used. Such separation methods include any suitable method known in the art (e.g., gelcaps, encapsulation, tablets, physical separation, etc.).

[0125] The cleaning compositions described herein are advantageously employed for example, in laundry applications, hard surface cleaning, dishwashing applications, as well as cosmetic applications such as dentures, teeth, hair, and skin. In addition, due to the unique advantages of increased effectiveness in lower temperature solutions, the Bsp Man4 enzymes described herein are ideally suited for laundry and fabric softening applications. Furthermore, the Bsp Man4 enzymes may find use in granular and liquid compositions.

[0126] The isolated Bsp Man4 polypeptides described herein may also find use cleaning in additive products. In some embodiments, low temperature solution cleaning applications find use. In some embodiments, the present disclosure provides cleaning additive products including at least one disclosed Bsp Man4 polypeptide is ideally suited for inclusion in a wash process when additional bleaching effectiveness is desired. Such instances include, but are not limited to low temperature solution cleaning applications. In some embodiments, the additive product is in its simplest form, one or more endo-.beta.-mannanases. In some embodiments, the additive is packaged in dosage form for addition to a cleaning process. In some embodiments, the additive is packaged in dosage form for addition to a cleaning process where a source of peroxygen is employed and increased bleaching effectiveness is desired. Any suitable single dosage unit form finds use with the present disclosure, including but not limited to pills, tablets, gelcaps, or other single dosage units such as pre-measured powders or liquids. In some embodiments, filler(s) or carrier material(s) are included to increase the volume of such compositions. Suitable filler or carrier materials include, but are not limited to various salts of sulfate, carbonate, and silicate as well as talc, clay, and the like. Suitable filler or carrier materials for liquid compositions include, but are not limited to water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to methanol, ethanol, propanol, and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials. Acidic fillers find use to reduce pH. Alternatively, in some embodiments, the cleaning additive includes adjunct ingredients, as described more fully below.

[0127] The present cleaning compositions and cleaning additives require an effective amount of at least one of the Bsp Man4 polypeptides described herein, alone or in combination with other endo-.beta.-mannanases and/or additional enzymes. In certain embodiments, the additional enzymes include, but are not limited to, at least one enzyme selected from proteases, peroxidases, cellulases (endoglucanases), beta-glucanases, hemicellulases, lipases, acyl transferases, phospholipases, esterases, laccases, catalases, aryl esterases, amylases, alpha-amylases, glucoamylases, cutinases, pectinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenases, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, other endo-.beta.-mannanases, exo-.beta.-mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, and mixtures thereof.

[0128] The required level of enzyme is achieved by the addition of one or more disclosed Bsp Man4 polypeptide. Typically the present cleaning compositions will comprise at least about 0.0001 weight percent, from about 0.0001 to about 10, from about 0.001 to about 1, or even from about 0.01 to about 0.1 weight percent of at least one of the disclosed Bsp Man4 polypeptides.

[0129] The cleaning compositions herein are typically formulated such that, during use in aqueous cleaning operations, the wash water will have a pH of from about 3.0 to about 11.0. Liquid product formulations are typically formulated to have a neat pH from about 5.0 to about 9.0. Granular laundry products are typically formulated to have a pH from about 8.0 to about 11.0. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0130] Suitable low pH cleaning compositions typically have a neat pH of from about 3.0 to about 5.0, or even a neat pH of from 3.5 to 4.5. Low pH cleaning compositions are typically free of surfactants that hydrolyze in such a pH environment. Such surfactants include sodium alkyl sulfate surfactants that comprise at least one ethylene oxide moiety or even from about 1 to about 16 moles of ethylene oxide. Such cleaning compositions typically comprise a sufficient amount of a pH modifier, such as sodium hydroxide, monoethanolamine, or hydrochloric acid, to provide such cleaning composition with a neat pH of from about 3.0 to about 5.0. Such compositions typically comprise at least one acid stable enzyme. In some embodiments, the compositions are liquids, while in other embodiments, they are solids. The pH of such liquid compositions is typically measured as a neat pH. The pH of such solid compositions is measured as a 10% solids solution of said composition wherein the solvent is distilled water. In these embodiments, all pH measurements are taken at 20.degree. C., unless otherwise indicated.

[0131] Suitable high pH cleaning compositions typically have a neat pH of from about 9.0 to about 11.0, or even a net pH of from 9.5 to 10.5. Such cleaning compositions typically comprise a sufficient amount of a pH modifier, such as sodium hydroxide, monoethanolamine, or hydrochloric acid, to provide such cleaning composition with a neat pH of from about 9.0 to about 11.0. Such compositions typically comprise at least one base-stable enzyme. In some embodiments, the compositions are liquids, while in other embodiments, they are solids. The pH of such liquid compositions is typically measured as a neat pH. The pH of such solid compositions is measured as a 10% solids solution of said composition wherein the solvent is distilled water. In these embodiments, all pH measurements are taken at 20.degree. C., unless otherwise indicated.

[0132] In some embodiments, when the Bsp Man4 polypeptide is employed in a granular composition or in a liquid, it is desirable for the Bsp Man4 polypeptide to be in the form of an encapsulated particle to protect the Bsp Man4 polypeptide from other components of the granular composition during storage. In addition, encapsulation is also a means of controlling the availability of the Bsp Man4 polypeptide during the cleaning process. In some embodiments, encapsulation enhances the performance of the Bsp Man4 polypeptide and/or additional enzymes. In this regard, the Bsp Man4 polypeptides of the present disclosure are encapsulated with any suitable encapsulating material known in the art. In some embodiments, the encapsulating material typically encapsulates at least part of the catalyst for the Bsp Man4 polypeptides described herein. Typically, the encapsulating material is water-soluble and/or water-dispersible. In some embodiments, the encapsulating material has a glass transition temperature (Tg) of 0.degree. C. or higher. Glass transition temperature is described in more detail in the PCT application WO 97/11151. The encapsulating material is typically selected from consisting of carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes, and combinations thereof. When the encapsulating material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. In some typical embodiments, the encapsulating material is a starch (See, e.g., EP 0 922 499; U.S. Pat. No. 4,977,252; U.S. Pat. No. 5,354,559; and U.S. Pat. No. 5,935,826). In some embodiments, the encapsulating material is a microsphere made from plastic such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile, and mixtures thereof; commercially available microspheres that find use include, but are not limited to those supplied by EXPANCEL.RTM. (Stockviksverken, Sweden), and PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES.RTM., LUXSIL.RTM., Q-CEL.RTM., and SPHERICEL.RTM. (PQ Corp., Valley Forge, Pa.).

[0133] The term "granular composition" refers to a conglomeration of discrete solid, macroscopic particles. Powders are a special class of granular material due to their small particle size, which makes them more cohesive and more easily suspended.

[0134] In using detergent compositions that include Bsp Man4 in cleaning applications, the fabrics, textiles, dishes, or other surfaces to be cleaned are incubated in the presence of the Bsp Man4 detergent composition for a time sufficient to allow Bsp Man4 to hydrolyze mannan substrates including, but not limited to, locust bean gum, guar gum, and combinations thereof present in soil or stains, and then typically rinsed with water or another aqueous solvent to remove the Bsp Man4 detergent composition along with hydrolyzed mannans.

[0135] As described herein, the Bsp Man4 polypeptides find particular use in the cleaning industry, including, but not limited to laundry and dish detergents. These applications place enzymes under various environmental stresses. The Bsp Man4 polypeptides may provide advantages over many currently used enzymes, due to their stability under various conditions.

[0136] Indeed, there are a variety of wash conditions including varying detergent formulations, wash water volumes, wash water temperatures, and lengths of wash time, to which endo-.beta.-mannanases involved in washing are exposed. In addition, detergent formulations used in different geographical areas have different concentrations of their relevant components present in the wash water. For example, European detergents typically have about 4500-5000 ppm of detergent components in the wash water, while Japanese detergents typically have approximately 667 ppm of detergent components in the wash water. In North America, particularly the United States, detergents typically have about 975 ppm of detergent components present in the wash water.

[0137] A low detergent concentration system includes detergents where less than about 800 ppm of the detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration system as they have approximately 667 ppm of detergent components present in the wash water.

[0138] A medium detergent concentration includes detergents where between about 800 ppm and about 2000 ppm of the detergent components are present in the wash water. North American detergents are generally considered to be medium detergent concentration systems as they have approximately 975 ppm of detergent components present in the wash water. Brazil typically has approximately 1500 ppm of detergent components present in the wash water.

[0139] A high detergent concentration system includes detergents where greater than about 2000 ppm of the detergent components are present in the wash water. European detergents are generally considered to be high detergent concentration systems as they have approximately 4500-5000 ppm of detergent components in the wash water.

[0140] Latin American detergents are generally high suds phosphate builder detergents and the range of detergents used in Latin America can fall in both the medium and high detergent concentrations as they range from 1500 ppm to 6000 ppm of detergent components in the wash water. As mentioned above, Brazil typically has approximately 1500 ppm of detergent components present in the wash water. However, other high suds phosphate builder detergent geographies, not limited to other Latin American countries, may have high detergent concentration systems up to about 6000 ppm of detergent components present in the wash water.

[0141] In light of the foregoing, it is evident that concentrations of detergent compositions in typical wash solutions throughout the world varies from less than about 800 ppm of detergent composition ("low detergent concentration geographies"), for example about 667 ppm in Japan, to between about 800 ppm to about 2000 ppm ("medium detergent concentration geographies"), for example about 975 ppm in U.S. and about 1500 ppm in Brazil, to greater than about 2000 ppm ("high detergent concentration geographies"), for example about 4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high suds phosphate builder geographies.

[0142] The concentrations of the typical wash solutions are determined empirically. For example, in the U.S., a typical washing machine holds a volume of about 64.4 L of wash solution. Accordingly, in order to obtain a concentration of about 975 ppm of detergent within the wash solution about 62.79 g of detergent composition must be added to the 64.4 L of wash solution. This amount is the typical amount measured into the wash water by the consumer using the measuring cup provided with the detergent.

[0143] As a further example, different geographies use different wash temperatures. The temperature of the wash water in Japan is typically less than that used in Europe. For example, the temperature of the wash water in North America and Japan is typically between about 10 and about 30.degree. C. (e.g., about 20.degree. C.), whereas the temperature of wash water in Europe is typically between about 30 and about 60.degree. C. (e.g., about 40.degree. C.). Accordingly, in certain embodiments, the detergent compositions described herein may be utilized at temperature from about 10.degree. C. to about 60.degree. C., or from about 20.degree. C. to about 60.degree. C., or from about 30.degree. C. to about 60.degree. C., or from about 40.degree. C. to about 60.degree. C., as well as all other combinations within the range of about 40.degree. C. to about 55.degree. C., and all ranges within 10.degree. C. to 60.degree. C. However, in the interest of saving energy, many consumers are switching to using cold water washing. In addition, in some further regions, cold water is typically used for laundry, as well as dish washing applications. In some embodiments, the "cold water washing" of the present disclosure utilizes washing at temperatures from about 10.degree. C. to about 40.degree. C., or from about 20.degree. C. to about 30.degree. C., or from about 15.degree. C. to about 25.degree. C., as well as all other combinations within the range of about 15.degree. C. to about 35.degree. C., and all ranges within 10.degree. C. to 40.degree. C.

[0144] As a further example, different geographies typically have different water hardness. Water hardness is usually described in terms of the grains per gallon mixed Ca.sup.2+/Mg.sup.2+. Hardness is a measure of the amount of calcium (Ca.sup.2+) and magnesium (Mg.sup.2+) in the water. Most water in the United States is hard, but the degree of hardness varies. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 parts per million (parts per million converted to grains per U.S. gallon is ppm # divided by 17.1 equals grains per gallon) of hardness minerals.

TABLE-US-00002 TABLE II Water Hardness Levels Water Grains per gallon Parts per million Soft less than 1.0 less than 17 Slightly hard 1.0 to 3.5 17 to 60 Moderately hard 3.5 to 7.0 60 to 120 Hard 7.0 to 10.5 120 to 180 Very hard greater than 10.5 greater than 180

[0145] European water hardness is typically greater than about 10.5 (for example about 10.5 to about 20.0) grains per gallon mixed Ca.sup.2+/Mg.sup.2+ (e.g., about 15 grains per gallon mixed Ca.sup.2+/Mg.sup.2+). North American water hardness is typically greater than Japanese water hardness, but less than European water hardness. For example, North American water hardness can be between about 3 to about 10 grains, about 3 to about 8 grains or about 6 grains. Japanese water hardness is typically lower than North American water hardness, usually less than about 4, for example about 3 grains per gallon mixed Ca.sup.2+/Mg.sup.2+.

[0146] Accordingly, in some embodiments, the present disclosure provides Bsp Man4 polypeptides that show surprising wash performance in at least one set of wash conditions (e.g., water temperature, water hardness, and/or detergent concentration). In some embodiments, the Bsp Man4 polypeptides are comparable in wash performance to other endo-.beta.-mannanases. In some embodiments, the Bsp Man4 polypeptides exhibit enhanced wash performance as compared to endo-.beta.-mannanases currently commercially available. Thus, in some preferred embodiments, the Bsp Man4 polypeptides provided herein exhibit enhanced oxidative stability, enhanced thermal stability, enhanced cleaning capabilities under various conditions, and/or enhanced chelator stability. In addition, the Bsp Man4 polypeptides may find use in cleaning compositions that do not include detergents, again either alone or in combination with builders and stabilizers.

[0147] In some embodiments of the present disclosure, the cleaning compositions comprise at least one Bsp Man4 polypeptide of the present disclosure at a level from about 0.00001% to about 10% by weight of the composition and the balance (e.g., about 99.999% to about 90.0%) comprising cleaning adjunct materials by weight of composition. In other aspects of the present disclosure, the cleaning compositions comprises at least one Bsp Man4 polypeptide at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% by weight of the composition and the balance of the cleaning composition (e.g., about 99.9999% to about 90.0%, about 99.999% to about 98%, about 99.995% to about 99.5% by weight) comprising cleaning adjunct materials.

[0148] In addition to the Bsp Man4 polypeptides provided herein, any other suitable endo-.beta.-mannanases find use in the compositions of the present disclosure. Suitable endo-.beta.-mannanases include, but are not limited to, endo-.beta.-mannanases of the GH26 family of glycosyl hydrolases, endo-.beta.-mannanases of the GH5 family of glycosyl hydrolases, acidic endo-.beta.-mannanases, neutral endo-.beta.-mannanases, and alkaline endo-.beta.-mannanases. Examples of alkaline endo-.beta.-mannanases include those described in U.S. Pat. Nos. 6,060,299, 6,566,114, and 6,602,842; WO 9535362A1, WO 9964573A1, and WO9964619A1. Additionally, suitable endo-.beta.-mannanases include, but are not limited to those of animal, plant, fungal, or bacterial origin. Chemically or genetically modified mutants are encompassed by the present disclosure.

[0149] Examples of useful endo-.beta.-mannanases include Bacillus endo-.beta.-mannanases such as B. subtilis endo-.beta.-mannanase (See, e.g., U.S. Pat. No. 6,060,299, and WO 9964573A1), B. sp. 1633 endo-.beta.-mannanase (See, e.g., U.S. Pat. No. 6,566,114 and WO9964619A1), Bacillus sp. AAI12 endo-.beta.-mannanase (See, e.g., U.S. Pat. No. 6,566,114 and WO9964619A1), B. sp. AA349 endo-.beta.-mannanase (See, e.g., U.S. Pat. No. 6,566,114 and WO9964619A1), B. agaradhaerens NCIMB 40482 endo-.beta.-mannanase (See, e.g., U.S. Pat. No. 6,566,114 and WO9964619A1), B. halodurans endo-.beta.-mannanase, B. clausii endo-.beta.-mannanase (See, e.g., U.S. Pat. No. 6,566,114 and WO9964619A1), B. licheniformis endo-.beta.-mannanase (See, e.g., U.S. Pat. No. 6,566,114 and WO9964619A1), Humicola endo-.beta.-mannanases such as H. insolens endo-.beta.-mannanase (See, e.g., U.S. Pat. No. 6,566,114 and WO9964619A1), and Caldocellulosiruptor endo-.beta.-mannanases such as C. sp. endo-.beta.-mannanase (See, e.g., U.S. Pat. No. 6,566,114 and WO9964619A1).

[0150] Furthermore, a number of identified mannanases (i.e., endo-.beta.-mannanases and exo-.beta.-mannanases) find use in some embodiments of the present disclosure, including but not limited to Agaricus bisporus mannanase (See, Tang et al., [2001] Appl. Environ. Microbiol. 67: 2298-2303), Aspergillu tamarii mannanase (See, Civas et al., [1984] Biochem. J. 219: 857-863), Aspergillus aculeatus mannanase (See, Christgau et al., [1994] Biochem. Mol. Biol. Int. 33: 917-925), Aspergillus awamori mannanase (See, Setati et al., [2001] Protein Express Purif. 21: 105-114), Aspergillus fumigatus mannanase (See, Puchart et al., [2004] Biochimica et biophysica Acta. 1674: 239-250), Aspergillus niger mannanase (See, Ademark et al., [1998] J. Biotechnol. 63: 199-210), Aspergillus oryzae NRRL mannanase (See, Regalado et al., [2000] J. Sci. Food Agric. 80: 1343-1350), Aspergillus sulphureus mannanase (See, Chen et al., [2007] J. Biotechnol. 128(3): 452-461), Aspergillus terrus mannanase (See, Huang et al., [2007] Wei Sheng Wu Xue Bao. 47(2): 280-284), Bacillus agaradhaerens mannanase (See, U.S. Pat. No. 6,376,445.), Bacillus AM001 mannanase (See, Akino et al., [1989] Arch. Microbiol. 152: 10-15), Bacillus brevis mannanase (See, Araujo and Ward, [1990] J. Appl. Bacteriol. 68: 253-261), Bacillus circulans K-1 mannanase (See, Yoshida et al., [1998] Biosci. Biotechnol. Biochem. 62(3): 514-520), Bacillus polymyxa mannanase (See, Araujo and Ward, [1990] J. Appl. Bacteriol. 68: 253-261), Bacillus sp JAMB-750 mannanase (See, Hatada et al., [2005] Extremophiles. 9: 497-500), Bacillus sp. M50 mannanase (See, Chen et al., [2000] Wei Sheng Wu Xue Bao. 40: 62-68), Bacillus sp. N 16-5 mannanase (See, Yanhe et al., [2004] Extremophiles 8: 447-454), Bacillus stearothermophilu mannanase (See, Talbot and Sygusch, [1990] Appl. Environ. Microbiol. 56: 3505-3510), Bacillus subtilis mannanase (See, Mendoza et al., [1994] World J. Microbiol. Biotechnol. 10: 51-54), Bacillus subtilis B36 mannanase (Li et al., [2006] Z. Naturforsch (C). 61: 840-846), Bacillus subtilis BM9602 mannanase (See, Cui et al., [1999] Wei Sheng Wu Xue Bao. 39(1): 60-63), Bacillus subtilis SA-22 mannanase (See, Sun et al., [2003] Sheng Wu Gong Cheng Xue Bao. 19(3): 327-330), Bacillus subtilis 168 mannanase (See, Helow and Khattab, [1996] Acta Microbiol. Immunol. Hung. 43: 289-299), Bacteroides ovatus mannanase (See, Gherardini et al., [1987] J. Bacteriol. 169: 2038-2043), Bacteroides ruminicola mannanase (See, Matsushita et al., [1991] J. Bacteriol. 173: 6919-6926), Caldibacillus cellulovorans mannanase (See, Sunna et al., [2000] Appl. Environ. Microbiol. 66: 664-670), Caldocellulosiruptor saccharolyticus mannanase (See, Morris et al., [1995]Appl. Environ. Microbiol. 61: 2262-2269), Caldocellum saccharolyticum mannanase (See, Bicho et al., [1991] Appl. Microbiol. Biotechnol. 36: 337-343), Cellulomonas fimi mannanase (See, Stoll et al., [1999] Appl. Environ. Microbiol. 65(6):2598-2605), Clostridium butyricum/beijerinckii mannanase (See, Nakajima and Matsuura, [1997] Biosci. Biotechnol. Biochem. 61: 1739-1742), Clostridium cellulolyticum mannanase (See, Perret et al., [2004] Biotechnol. Appl. Biochem. 40: 255-259), Clostridium tertium mannanase (See, Kataoka and Tokiwa, [1998] J. Appl. Microbiol. 84: 357-367), Clostridium thermocellum mannanase (See, Halstead et al., [1999] Microbiol. 145: 3101-3108), Dictyoglomus thermophilum mannanase (See, Gibbs et al., [1999] Curr. Microbiol. 39(6): 351-357), Flavobacterium sp mannanase (See, Zakaria et al., [1998] Biosci. Biotechnol. Biochem. 62: 655-660), Gastropoda pulmonata mannanase (See, Charrier and Rouland, [2001] J. Expt. Zool. 290: 125-135), Littorina brevicula mannanase (See, Yamamura et al., [1996] Biosci. Biotechnol. Biochem. 60: 674-676), Lycopersicon esculentum mannanase (See, Filichkin et al., [2000] Plant Physiol. 134:1080-1087), Paenibacillus curdlanolyticus mannanase (See, Pason and Ratanakhanokchai, [2006] Appl. Environ. Microbiol. 72: 2483-2490), Paenibacillus polymyxa mannanase (See, Han et al., [2006] Appl. Microbiol. Biotechnol. 73(3): 618-630), Phanerochaete chrysosporium mannanase (See, Wymelenberg et al., [2005] J. Biotechnol. 118: 17-34), Piromyces sp. mannanase (See, Fanutti et al., [1995] J. Biol. Chem. 270(49): 29314-29322), Pomacea insulars mannanase (See, Yamamura et al., [1993] Biosci. Biotechnol. Biochem. 7: 1316-1319), Pseudomonas fluorescens sub sp. Cellulose mannanase (See, Braithwaite et al., [1995] Biochem J. 305: 1005-1010), Rhodothermus marinus mannanase (See, Politz et al., [2000] Appl. Microbiol. Biotechnol. 53 (6): 715-721), Sclerotium rolfsii mannanase (See, Sachslehner et al., [2000] J. Biotechnol. 80:127-134), Streptomyces galbus mannanase (See, Kansoh and Nagieb, [2004] Anton. van. Leeuwonhoek. 85: 103-114), Streptomyces lividans mannanase (See, Arcand et al., [1993] J. Biochem. 290: 857-863), Thermoanaerobacterium Polysaccharolyticum mannanase (See, Cann et al., [1999] J. Bacteriol. 181: 1643-1651), Thermomonospora fusca mannanase (See, Hilge et al., [1998] Structure 6: 1433-1444), Thermotoga maritima mannanase (See, Parker et al., [2001] Biotechnol. Bioeng. 75(3): 322-333), Thermotoga neapolitana mannanase (See, Duffaud et al., [1997] Appl. Environ. Microbiol. 63: 169-177), Trichoderma harzanium strain T4 mannanase (See, Franco et al., [2004] Biotechnol Appl. Biochem. 40: 255-259), Trichoderma reesei mannanase (See, Stalbrand et al., [1993] J. Biotechnol. 29: 229-242), and Vibrio sp. mannanase (See, Tamaru et al., [1997] J. Ferment. Bioeng. 83: 201-205).

[0151] Additional suitable endo-.beta.-mannanases include commercially available endo-.beta.-mannanases such as HEMICELL.RTM. (Chemgen); GAMANASE.RTM. and MANNAWAY.RTM., (Novozymes A/S, Denmark); PURABRITE.TM. and MANNASTAR.TM. (Genencor, A Danisco Division, Palo Alto, Calif.); and PYROLASE.RTM. 160 and PYROLASE.RTM. 200 (Diversa).

[0152] In some embodiments of the present disclosure, the cleaning compositions of the present disclosure further comprise endo-.beta.-mannanases at a level from about 0.00001% to about 10% of additional endo-.beta.-mannanase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present disclosure, the cleaning compositions of the present disclosure also comprise endo-.beta.-mannanases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% endo-.beta.-mannanase by weight of the composition.

[0153] In some embodiments of the present disclosure, any suitable protease may be used. Suitable proteases include those of animal, vegetable or microbial origin. In some embodiments, chemically or genetically modified mutants are included. In some embodiments, the protease is a serine protease, preferably an alkaline microbial protease or a trypsin-like protease. Various proteases are described in PCT applications WO 95/23221 and WO 92/21760; U.S. Pat. Publication No. 2008/0090747; and U.S. Pat. Nos. 5,801,039; 5,340,735; 5,500,364; 5,855,625; U.S. RE 34,606; U.S. Pat. Nos. 5,955,340; 5,700,676; 6,312,936; 6,482,628; and various other patents. In some further embodiments, metalloproteases find use in the present disclosure, including but not limited to the neutral metalloprotease described in PCT application WO 07/044,993. Commercially available proteases that find use in the present disclosure include, but are not limited to PURAFECT.RTM., PURAFECT.RTM. PRIME, and PROPERASE.RTM. (Genencor, A Danisco Division, Palo Alto, Calif.). Additionally, commercially available proteases that find use in the present disclosure include, but are not limited to ALCALASE.RTM., EVERLASE.RTM., LIQUINASE.RTM., POLARZYME.RTM., OVOZYME.RTM. and SAVINASE.RTM. (Novozymes A/S, Denmark).

[0154] In some embodiments of the present disclosure, any suitable amylase may be used. In some embodiments, any amylase (e.g., alpha and/or beta) suitable for use in alkaline solutions also find use. Suitable amylases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments. Amylases that find use in the present disclosure include, but are not limited to .alpha.-amylases obtained from B. licheniformis (See, e.g., GB 1,296,839). Commercially available amylases that find use in the present disclosure include, but are not limited to DURAMYL.RTM., TERMAMYL.RTM., FUNGAMYL.RTM., STAINZYME.RTM., STAINZYME PLUS.RTM., STAINZYME ULTRA.RTM., and BAN.TM. (Novozymes A/S, Denmark), as well as PURASTAR.RTM., POWERASE.TM., RAPIDASE.RTM., and MAXAMYL.RTM. P (Genencor, A Danisco Division, Palo Alto, Calif.).

[0155] In some embodiments of the present disclosure, the disclosed cleaning compositions further comprise amylases at a level from about 0.00001% to about 10% of additional amylase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present disclosure, the cleaning compositions also comprise amylases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% amylase by weight of the composition.

[0156] In some embodiments of the present disclosure, any suitable pectin degrading enzyme may be used. As used herein, "pectin degrading enzyme(s)" encompass arabinanase (EC 3.2.1.99), galactanases (EC 3.2.1.89), polygalacturonase (EC 3.2.1.15) exo-polygalacturonase (EC 3.2.1.67), exo-poly-alpha-galacturonidase (EC 3.2.1.82), pectin lyase (EC 4.2.2.10), pectin esterase (EC 3.2.1.11), pectate lyase (EC 4.2.2.2), exo-polygalacturonate lyase (EC 4.2.2.9) and hemicellulases such as endo-1,3-.beta.-xylosidase (EC 3.2.1.32), xylan-1,4-.beta.-xylosidase (EC 3.2.1.37) and .alpha.-L-arabinofuranosidase (EC 3.2.1.55). Pectin degrading enzymes are natural mixtures of the above mentioned enzymatic activities. Pectin enzymes therefore include the pectin methylesterases which hdyrolyse the pectin methyl ester linkages, polygalacturonases which cleave the glycosidic bonds between galacturonic acid molecules, and the pectin transeliminases or lyases which act on the pectic acids to bring about non-hydrolytic cleavage of .alpha.-1,4 glycosidic linkages to form unsaturated derivatives of galacturonic acid.

[0157] Suitable pectin degrading enzymes include those of plant, fungal, or microbial origin. In some embodiments, chemically or genetically modified mutants are included. In some embodiments, the pectin degrading enzymes are alkaline pectin degrading enzymes, i.e., enzymes having an enzymatic activity of at least 10%, preferably at least 25%, more preferably at least 40% of their maximum activity at a pH of from about 7.0 to about 12. In certain other embodiments, the pectin degrading enzymes are enzymes having their maximum activity at a pH of from about 7.0 to about 12. Alkaline pectin degrading enzymes are produced by alkalophilic microorganisms e.g., bacterial, fungal, and yeast microorganisms such as Bacillus species. In some embodiments, the microorganisms are Bacillus firmus, Bacillus circulans, and Bacillus subtilis as described in JP 56131376 and JP 56068393. Alkaline pectin decomposing enzymes may include but are not limited to galacturn-1,4-.alpha.-galacturonase (EC 3.2.1.67), poly-galacturonase activities (EC 3.2.1.15, pectin esterase (EC 3.1.1.11), pectate lyase (EC 4.2.2.2) and their iso enzymes. Alkaline pectin decomposing enzymes can be produced by the Erwinia species. In some embodiments, the alkaline pectin decomposing enzymes are produced by E. chrysanthemi, E. carotovora, E. amylovora, E. herbicola, and E. dissolvens as described in JP 59066588, JP 63042988, and in World J. Microbiol. Microbiotechnol. (8, 2, 115-120) 1992. In certain other embodiments, the alkaline pectin enzymes are produced by Bacillus species as disclosed in JP 73006557 and Agr. Biol. Chem. (1972), 36 (2) 285-93.

[0158] In some embodiments of the present disclosure, the disclosed cleaning compositions further comprise pectin degrading enzymes at a level from about 0.00001% to about 10% of additional pectin degrading enzyme by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present disclosure, the cleaning compositions also comprise pectin degrading enzymes at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% pectin degrading enzyme by weight of the composition.

[0159] In some other embodiments, any suitable xyloglucanase finds used in the cleaning compositions of the present disclosure. Suitable xyloglucanases include, but are not limited to those of plant, fungal, or bacterial origin. Chemically or genetically modified mutants are included in some embodiments. As used herein, "xyloglucanase(s)" encompass the family of enzymes described by Vincken and Voragen at Wageningen University [Vincken et al (1994) Plant Physiol., 104, 99-107] and are able to degrade xyloglucans as described in Hayashi et al (1989) Plant. Physiol. Plant Mol. Biol., 40, 139-168. Vincken et al demonstrated the removal of xyloglucan coating from cellulose of the isolated apple cell wall by a xyloglucanase purified from Trichoderma viride (endo-IV-glucanase). This enzyme enhances the enzymatic degradation of cell wall-embedded cellulose and work in synergy with pectic enzymes. Rapidase LIQ+ from Gist-Brocades contains a xyloglucanase activity.

[0160] In some embodiments of the present disclosure, the disclosed cleaning compositions further comprise xyloglucanases at a level from about 0.00001% to about 10% of additional xyloglucanase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present disclosure, the cleaning compositions also comprise xyloglucanases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% xyloglucanase by weight of the composition. In certain other embodiments, xyloglucanases for specific applications are alkaline xyloglucanases, i.e., enzymes having an enzymatic activity of at least 10%, preferably at lest 25%, more preferably at least 40% of their maximum activity at a pH ranging from 7 to 12. In certain other embodiments, the xyloglucanases are enzymes having their maximum activity at a pH of from about 7.0 to about 12.

[0161] In some further embodiments, any suitable cellulase finds used in the cleaning compositions of the present disclosure. Suitable cellulases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments. Suitable cellulases include, but are not limited to Humicola insolens cellulases (See, e.g., U.S. Pat. No. 4,435,307). Especially suitable cellulases are the cellulases having color care benefits (See, e.g., EP 0 495 257). Commercially available cellulases that find use in the present disclosure include, but are not limited to ENDOLASE.RTM., CELLUCLEAN.RTM., CELLUZYME.RTM., CAREZYME.RTM. (Novozymes A/S, Denmark). Additional commercially available cellulases include PURADEX.RTM. (Genencor, A Danisco Division, Palo Alto, Calif.) and KAC-500(B).TM. (Kao Corporation). In some embodiments, cellulases are incorporated as portions or fragments of mature wild-type or variant cellulases, wherein a portion of the N-terminus is deleted (See, e.g., U.S. Pat. No. 5,874,276). In some embodiments, the cleaning compositions of the present disclosure further comprise cellulases at a level from about 0.00001% to about 10% of additional cellulase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present disclosure, the cleaning compositions also comprise cellulases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% cellulase by weight of the composition.

[0162] In still further embodiments, any lipase suitable for use in detergent compositions also finds use in the present disclosure. Suitable lipases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments. Examples of useful lipases include Humicola lanuginosa lipase (See, e.g., EP 258 068, and EP 305 216), Rhizomucor miehei lipase (See, e.g., EP 238 023), Candida lipase, such as C. antarctica lipase (e.g., the C. antarctica lipase A or B; see, e.g., EP 214 761), Pseudomonas lipases such as P. alcaligenes lipase and P. pseudoalcaligenes lipase (See, e.g., EP 218 272), P. cepacia lipase (See, e.g., EP 331 376), P. stutzeri lipase (See, e.g., GB 1,372,034), P. fluorescens lipase, Bacillus lipase (e.g., B. subtilis lipase [Dartois et al., (1993) Biochem. Biophys. Acta 1131:253-260]; B. stearothermophilus lipase [See, e.g., JP 64/744992]; and B. pumilus lipase [See, e.g., WO 91/16422]). Furthermore, a number of cloned lipases find use in some embodiments of the present disclosure, including but not limited to Penicillium camembertii lipase (See, Yamaguchi et al., [1991] Gene 103:61-67), Geotricum candidum lipase (See, Schimada et al., [1989] J. Biochem. 106:383-388), and various Rhizopus lipases such as R. delemar lipase (See, Hass et al., [1991] Gene 109:117-113), R. niveus lipase (Kugimiya et al., [1992] Biosci. Biotech. Biochem. 56:716-719), and R. oryzae lipase. Other types of lipolytic enzymes such as cutinases also find use in some embodiments of the present disclosure, including but not limited to the cutinase derived from Pseudomonas mendocina (See, WO 88/09367), and the cutinase derived from Fusarium solani pisi (See, WO 90/09446). Additional suitable lipases include commercially available lipases such as M1 LIPASE.TM., LUMA FAST.TM., and LIPOMAX.TM. (Genencor, A Danisco Division, Palo Alto, Calif.); LIPEX.RTM., LIPOCLEAN.RTM., LIPOLASE.RTM. and LIPOLASE.RTM. ULTRA (Novozymes A/S, Denmark); and LIPASE P.TM."Amano" (Amano Pharmaceutical Co. Ltd., Japan).

[0163] In some embodiments, the disclosed cleaning compositions further comprise lipases at a level from about 0.00001% to about 10% of additional lipase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present disclosure, the cleaning compositions also comprise lipases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% lipase by weight of the composition.

[0164] In some embodiments, peroxidases are used in combination with hydrogen peroxide or a source thereof (e.g., a percarbonate, perborate or persulfate) in the compositions of the present disclosure. In some alternative embodiments, oxidases are used in combination with oxygen. Both types of enzymes are used for "solution bleaching" (i.e., to prevent transfer of a textile dye from a dyed fabric to another fabric when the fabrics are washed together in a wash liquor), preferably together with an enhancing agent (See, e.g., WO 94/12621 and WO 95/01426). Suitable peroxidases/oxidases include, but are not limited to those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments. In some embodiments, the cleaning compositions of the present disclosure further comprise peroxidase and/or oxidase enzymes at a level from about 0.00001% to about 10% of additional peroxidase and/or oxidase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present disclosure, the cleaning compositions also comprise peroxidase and/or oxidase enzymes at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% peroxidase and/or oxidase enzymes by weight of the composition.

[0165] In some embodiments, additional enzymes find use, including but not limited to perhydrolases (See, e.g., WO 05/056782). In addition, in some particularly preferred embodiments, mixtures of the above mentioned enzymes are encompassed herein, in particular one or more additional protease, amylase, lipase, mannanase, and/or at least one cellulase. Indeed, it is contemplated that various mixtures of these enzymes will find use in the present disclosure. It is also contemplated that the varying levels of the Bsp Man4 polypeptide(s) and one or more additional enzymes may both independently range to about 10%, the balance of the cleaning composition being cleaning adjunct materials. The specific selection of cleaning adjunct materials are readily made by considering the surface, item, or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use (e.g., through the wash detergent use).

[0166] Examples of suitable cleaning adjunct materials include, but are not limited to, surfactants, builders, bleaches, bleach activators, bleach catalysts, other enzymes, enzyme stabilizing systems, chelants, optical brighteners, soil release polymers, dye transfer agents, dye transfer inhibiting agents, catalytic materials, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal agents, structure elasticizing agents, dispersants, suds suppressors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescers, fabric conditioners, fabric softeners, carriers, hydrotropes, processing aids, solvents, pigments, hydrolyzable surfactants, preservatives, anti-oxidants, anti-shrinkage agents, anti-wrinkle agents, germicides, fungicides, color speckles, silvercare, anti-tarnish and/or anti-corrosion agents, alkalinity sources, solubilizing agents, carriers, processing aids, pigments, and pH control agents (See, e.g., U.S. Pat. Nos. 6,610,642; 6,605,458; 5,705,464; 5,710,115; 5,698,504; 5,695,679; 5,686,014; and 5,646,101; all of which are incorporated herein by reference). Embodiments of specific cleaning composition materials are exemplified in detail below. In embodiments in which the cleaning adjunct materials are not compatible with the disclosed Bsp Man4 polypeptides in the cleaning compositions, then suitable methods of keeping the cleaning adjunct materials and the endo-.beta.-mannanase(s) separated (i.e., not in contact with each other) until combination of the two components is appropriate are used. Such separation methods include any suitable method known in the art (e.g., gelcaps, encapsulation, tablets, physical separation, etc.).

[0167] In some preferred embodiments, an effective amount of one or more Bsp Man4 polypeptide(s) provided herein are included in compositions useful for cleaning a variety of surfaces in need of stain removal. Such cleaning compositions include cleaning compositions for such applications as cleaning hard surfaces, fabrics, and dishes. Indeed, in some embodiments, the present disclosure provides fabric cleaning compositions, while in other embodiments, the present disclosure provides non-fabric cleaning compositions. Notably, the present disclosure also provides cleaning compositions suitable for personal care, including oral care (including dentrifices, toothpastes, mouthwashes, etc., as well as denture cleaning compositions), skin, and hair cleaning compositions. Additionally, in still other embodiments, the present disclosure provides fabric softening compositions. It is intended that the present disclosure encompass detergent compositions in any form (i.e., liquid, granular, bar, semi-solid, gels, emulsions, tablets, capsules, etc.).

[0168] By way of example, several cleaning compositions wherein the disclosed Bsp Man4 polypeptides find use are described in greater detail below. In some embodiments in which the disclosed cleaning compositions are formulated as compositions suitable for use in laundry machine washing method(s), the compositions of the present disclosure preferably contain at least one surfactant and at least one builder compound, as well as one or more cleaning adjunct materials preferably selected from organic polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti-redeposition agents and corrosion inhibitors. In some embodiments, laundry compositions also contain softening agents (i.e., as additional cleaning adjunct materials). The compositions of the present disclosure also find use detergent additive products in solid or liquid form. Such additive products are intended to supplement and/or boost the performance of conventional detergent compositions and can be added at any stage of the cleaning process. In some embodiments, the density of the laundry detergent compositions herein ranges from about 400 to about 1200 g/liter, while in other embodiments, it ranges from about 500 to about 950 g/liter of composition measured at 20.degree. C.

[0169] In embodiments formulated as compositions for use in manual dishwashing methods, the compositions of the disclosure preferably contain at least one surfactant and preferably at least one additional cleaning adjunct material selected from organic polymeric compounds, suds enhancing agents, group II metal ions, solvents, hydrotropes, and additional enzymes.

[0170] In some embodiments, various cleaning compositions such as those provided in U.S. Pat. No. 6,605,458 find use with the Bsp Man4 polypeptides of the present disclosure. Thus, in some embodiments, the compositions comprising at least one Bsp Man4 polypeptide of the present disclosure is a compact granular fabric cleaning composition, while in other embodiments, the composition is a granular fabric cleaning composition useful in the laundering of colored fabrics, in further embodiments, the composition is a granular fabric cleaning composition which provides softening through the wash capacity, in additional embodiments, the composition is a heavy duty liquid fabric cleaning composition. In some embodiments, the compositions comprising at least one Bsp Man4 polypeptide of the present disclosure are fabric cleaning compositions such as those described in U.S. Pat. Nos. 6,610,642 and 6,376,450. In addition, the Bsp Man4 polypeptides of the present disclosure find use in granular laundry detergent compositions of particular utility under European or Japanese washing conditions (See, e.g., U.S. Pat. No. 6,610,642).

[0171] In some alternative embodiments, the present disclosure provides hard surface cleaning compositions comprising at least one Bsp Man4 polypeptide provided herein. Thus, in some embodiments, the compositions comprising at least one Bsp Man4 polypeptide of the present disclosure is a hard surface cleaning composition such as those described in U.S. Pat. Nos. 6,610,642; 6,376,450; and 6,376,450.

[0172] In yet further embodiments, the present disclosure provides dishwashing compositions comprising at least one Bsp Man4 polypeptide provided herein. Thus, in some embodiments, the compositions comprising at least one Bsp Man4 polypeptide of the present disclosure is a hard surface cleaning composition such as those in U.S. Pat. Nos. 6,610,642 and 6,376,450. In some still further embodiments, the present disclosure provides dishwashing compositions comprising at least one Bsp Man4 polypeptide provided herein. In some further embodiments, the compositions comprising at least one Bsp Man4 polypeptide of the present disclosure comprise oral care compositions such as those in U.S. Pat. Nos. 6,376,450 and 6,605,458. The formulations and descriptions of the compounds and cleaning adjunct materials contained in the aforementioned U.S. Pat. Nos. 6,376,450; 6,605,458; and 6,610,642 find use with the Bsp Man4 polypeptides provided herein.

[0173] In still further embodiments, the compositions comprising at least one Bsp Man4 polypeptide of the present disclosure comprise fabric softening compositions such as those in GB-A1 400898, GB-A1 514 276, EP 0 011 340, EP 0 026 528, EP 0 242 919, EP 0 299 575, EP 0 313 146, and U.S. Pat. No. 5,019,292. The formulations and descriptions of the compounds and softening agents contained in the aforementioned GB-A1 400898, GB-A1 514 276, EP 0 011 340, EP 0 026 528, EP 0 242 919, EP 0 299 575, EP 0 313 146, and U.S. Pat. No. 5,019,292 find use with the Bsp Man4 polypeptides provided herein

[0174] The cleaning compositions of the present disclosure are formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in U.S. Pat. Nos. 5,879,584; 5,691,297; 5,574,005; 5,569,645; 5,565,422; 5,516,448; 5,489,392; and 5,486,303; all of which are incorporated herein by reference. When a low pH cleaning composition is desired, the pH of such composition is adjusted via the addition of a material such as monoethanolamine or an acidic material such as HCl.

[0175] While not essential for the purposes of the present disclosure, the non-limiting list of adjuncts illustrated hereinafter are suitable for use in the instant cleaning compositions. In some embodiments, these adjuncts are incorporated for example, to assist or enhance cleaning performance, for treatment of the substrate to be cleaned, or to modify the aesthetics of the cleaning composition as is the case with perfumes, colorants, dyes or the like. It is understood that such adjuncts are in addition to the Bsp Man4 polypeptides of the present disclosure. The precise nature of these additional components, and levels of incorporation thereof, will depend on the physical form of the composition and the nature of the cleaning operation for which it is to be used. Suitable adjunct materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, additional enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids and/or pigments. In addition to the disclosure below, suitable examples of such other adjuncts and levels of use are found in U.S. Pat. Nos. 5,576,282; 6,306,812; and 6,326,348 are incorporated by reference. The aforementioned adjunct ingredients may constitute the balance of the cleaning compositions of the present disclosure.

[0176] In some embodiments, the cleaning compositions according to the present disclosure comprise at least one surfactant and/or a surfactant system wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. In some low pH cleaning composition embodiments (e.g., compositions having a neat pH of from about 3 to about 5), the composition typically does not contain alkyl ethoxylated sulfate, as it is believed that such surfactant may be hydrolyzed by such compositions' acidic contents. In some embodiments, the surfactant is present at a level of from about 0.1% to about 60%, while in alternative embodiments the level is from about 1% to about 50%, while in still further embodiments the level is from about 5% to about 40%, by weight of the cleaning composition.

[0177] In some embodiments, the cleaning compositions of the present disclosure contain at least one chelating agent. Suitable chelating agents may include, but are not limited to copper, iron, and/or manganese chelating agents, and mixtures thereof. In embodiments in which at least one chelating agent is used, the cleaning compositions of the present disclosure comprise from about 0.1% to about 15% or even from about 3.0% to about 10% chelating agent by weight of the subject cleaning composition.

[0178] In some still further embodiments, the cleaning compositions provided herein contain at least one deposition aid. Suitable deposition aids include, but are not limited to, polyethylene glycol, polypropylene glycol, polycarboxylate, soil release polymers such as polytelephthalic acid, clays such as kaolinite, montmorillonite, atapulgite, illite, bentonite, halloysite, and mixtures thereof.

[0179] As indicated herein, in some embodiments, anti-redeposition agents find use in some embodiments of the present disclosure. In some preferred embodiments, non-ionic surfactants find use. For example, in automatic dishwashing embodiments, non-ionic surfactants find use for surface modification purposes, in particular for sheeting, to avoid filming and spotting and to improve shine. These non-ionic surfactants also find use in preventing the re-deposition of soils. In some preferred embodiments, the anti-redeposition agent is a non-ionic surfactant as known in the art (See, e.g., EP 2 100 949).

[0180] In some embodiments, the cleaning compositions of the present disclosure include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones, and polyvinylimidazoles, or mixtures thereof. In embodiments in which at least one dye transfer inhibiting agent is used, the cleaning compositions of the present disclosure comprise from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% by weight of the cleaning composition.

[0181] In some embodiments, silicates are included within the compositions of the present disclosure. In some such embodiments, sodium silicates (e.g., sodium disilicate, sodium metasilicate, and crystalline phyllosilicates) find use. In some embodiments, silicates are present at a level of from about 1% to about 20%. In some preferred embodiments, silicates are present at a level of from about 5% to about 15% by weight of the composition.

[0182] In some still additional embodiments, the cleaning compositions of the present disclosure also contain dispersants. Suitable water-soluble organic materials include, but are not limited to the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.

[0183] In some further embodiments, the enzymes used in the cleaning compositions are stabilized by any suitable technique. In some embodiments, the enzymes employed herein are stabilized by the presence of water-soluble sources of calcium and/or magnesium ions in the finished compositions that provide such ions to the enzymes. In some embodiments, the enzyme stabilizers include oligosaccharides, polysaccharides, and inorganic divalent metal salts, including alkaline earth metals, such as calcium salts. It is contemplated that various techniques for enzyme stabilization will find use in the present disclosure. For example, in some embodiments, the enzymes employed herein are stabilized by the presence of water-soluble sources of zinc (II), calcium (II), and/or magnesium (II) ions in the finished compositions that provide such ions to the enzymes, as well as other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), tin (II), cobalt (II), copper (II), nickel (II), and oxovanadium (IV). Chlorides and sulfates also find use in some embodiments of the present disclosure. Examples of suitable oligosaccharides and polysaccharides (e.g., dextrins) are known in the art (See, e.g., WO 07/145,964). In some embodiments, reversible protease inhibitors also find use, such as boron-containing compounds (e.g., borate, 4-formyl phenyl boronic acid) and/or a tripeptide aldehyde find use to further improve stability, as desired.

[0184] In some embodiments, bleaches, bleach activators, and/or bleach catalysts are present in the compositions of the present disclosure. In some embodiments, the cleaning compositions of the present disclosure comprise inorganic and/or organic bleaching compound(s). Inorganic bleaches may include, but are not limited to perhydrate salts (e.g., perborate, percarbonate, perphosphate, persulfate, and persilicate salts). In some embodiments, inorganic perhydrate salts are alkali metal salts. In some embodiments, inorganic perhydrate salts are included as the crystalline solid, without additional protection, although in some other embodiments, the salt is coated. Any suitable salt known in the art finds use in the present disclosure (See, e.g., EP 2 100 949).

[0185] In some embodiments, bleach activators are used in the compositions of the present disclosure. Bleach activators are typically organic peracid precursors that enhance the bleaching action in the course of cleaning at temperatures of 60.degree. C. and below. Bleach activators suitable for use herein include compounds which, under perhydrolysis conditions, give aliphatic peroxycarboxylic acids having preferably from about 1 to about 10 carbon atoms, in particular from about 2 to about 4 carbon atoms, and/or optionally substituted perbenzoic acid. Additional bleach activators are known in the art and find use in the present disclosure (See, e.g., EP 2 100 949).

[0186] In addition, in some embodiments and as further described herein, the cleaning compositions of the present disclosure further comprise at least one bleach catalyst. In some embodiments, the manganese triazacyclononane and related complexes find use, as well as cobalt, copper, manganese, and iron complexes. Additional bleach catalysts find use in the present disclosure (See, e.g., U.S. Pat. No. 4,246,612; U.S. Pat. No. 5,227,084; U.S. Pat. No. 4,810,410; WO 99/06521; and EP 2 100 949).

[0187] In some embodiments, the cleaning compositions of the present disclosure contain one or more catalytic metal complexes. In some embodiments, a metal-containing bleach catalyst finds use. In some preferred embodiments, the metal bleach catalyst comprises a catalyst system comprising a transition metal cation of defined bleach catalytic activity, (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations), an auxiliary metal cation having little or no bleach catalytic activity (e.g., zinc or aluminum cations), and a sequestrate having defined stability constants for the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra (methylenephosphonic acid) and water-soluble salts thereof are used (See, e.g., U.S. Pat. No. 4,430,243). In some embodiments, the cleaning compositions of the present disclosure are catalyzed by means of a manganese compound. Such compounds and levels of use are well known in the art (See, e.g., U.S. Pat. No. 5,576,282). In additional embodiments, cobalt bleach catalysts find use in the cleaning compositions of the present disclosure. Various cobalt bleach catalysts are known in the art (See, e.g., U.S. Pat. Nos. 5,597,936 and 5,595,967) and are readily prepared by known procedures.

[0188] In some additional embodiments, the cleaning compositions of the present disclosure include a transition metal complex of a macropolycyclic rigid ligand (MRL). As a practical matter, and not by way of limitation, in some embodiments, the compositions and cleaning processes provided by the present disclosure are adjusted to provide on the order of at least one part per hundred million of the active MRL species in the aqueous washing medium, and in some preferred embodiments, provide from about 0.005 ppm to about 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, and most preferably from about 0.1 ppm to about 5 ppm, of the MRL in the wash liquor.

[0189] In some embodiments, preferred transition-metals in the instant transition-metal bleach catalyst include, but are not limited to manganese, iron, and chromium. Preferred MRLs also include, but are not limited to special ultra-rigid ligands that are cross-bridged (e.g., 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2] hexadecane). Suitable transition metal MRLs are readily prepared by known procedures (See, e.g., WO 2000/32601 and U.S. Pat. No. 6,225,464).

[0190] In some embodiments, the cleaning compositions of the present disclosure comprise metal care agents. Metal care agents find use in preventing and/or reducing the tarnishing, corrosion, and/or oxidation of metals, including aluminum, stainless steel, and non-ferrous metals (e.g., silver and copper). Suitable metal care agents include those described in EP 2 100 949, WO 94/26860, and WO 94/26859). In some embodiments, the metal care agent is a zinc salt. In some further embodiments, the cleaning compositions of the present disclosure comprise from about 0.1% to about 5% by weight of one or more metal care agent.

[0191] As indicated above, the cleaning compositions of the present disclosure are formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in U.S. Pat. Nos. 5,879,584; 5,691,297; 5,574,005; 5,569,645; 5,516,448; 5,489,392; and 5,486,303; all of which are incorporated herein by reference. In some embodiments in which a low pH cleaning composition is desired, the pH of such composition is adjusted via the addition of an acidic material such as HCl.

[0192] The cleaning compositions disclosed herein of find use in cleaning a situs (e.g., a surface, dishware, or fabric). Typically, at least a portion of the situs is contacted with an embodiment of the present cleaning composition, in neat form or diluted in wash liquor, and then the situs is optionally washed and/or rinsed. For purposes of the present disclosure, "washing" includes but is not limited to, scrubbing and mechanical agitation. In some embodiments, the cleaning compositions are typically employed at concentrations of from about 500 ppm to about 15,000 ppm in solution. When the wash solvent is water, the water temperature typically ranges from about 5.degree. C. to about 90.degree. C. and, when the situs comprises a fabric, the water to fabric mass ratio is typically from about 1:1 to about 30:1.

VI. Bsp Man4 Polypeptides as Chemical Reagents

[0193] The preference of Bsp Man4 for polysaccharide chains containing mannose units, including but not limited to mannans, galactomannans, and glucomannans, makes the present polypeptides particularly useful for performing mannan hydrolysis reactions involving polysaccharide substrates containing 1, 4-.beta.-D-mannosidic linkages.

[0194] In general terms, a donor molecule is incubated in the presence of an isolated Bsp Man4 polypeptide or fragment or variant thereof under conditions suitable for performing a mannan hydrolysis reaction, followed by, optionally, isolating a product from the reaction. Alternatively, in the context of a foodstuff, the product may become a component of the foodstuff without isolation. In certain embodiments, the donor molecule is a polysaccharide chain comprising mannose units, including but not limited to mannans, glucomannans, galactomannans, and galactoglucomannans.

VII. Bsp Man4 Polypeptides for Food Processing and Animal Feed

[0195] Several anti-nutritional factors can limit the use of specific plant material in the preparation of animal feed and food for humans. For example, plant material containing oligomannans such as mannan, galactomannan, glucomannan and galactoglucomannan can reduce the digestibility and absorption of nutritional compounds such as minerals, vitamins, sugars and fats by the animals. The negative effects are in particular due to the high viscosity of the mannan-containing polymers and to the ability of the mannan-containing polymers to adsorb nutritional compounds. These effects are reduced through the use of mannan-containing polymers degrading enzymes, namely endo-.beta.-mannanase enzymes such as the Bsp Man4 polypeptides described herein, which permit a higher proportion of mannan-containing polymers containing cheap plant material to be included in the feed resulting in a reduction of feed costs. Additionally, through the activity of the Bsp Man4 polypeptides, mannan-containing polymers are broken down to simpler sugars, which can be more readily assimilated to provide additional energy. Accordingly, compositions comprising any of the Bsp Man4 polypeptides described herein preferably used for processing and/or manufacturing of food or animal feed.

[0196] In one aspect of the invention, there is provided a bread improver composition comprising any of the Bsp Man4 polypeptides of the current invention, optionally with a source of mannan or glucomannan or galactomannan present, and further optionally with other enzymes present.

[0197] In general terms animal feed containing plant material is incubated in the presence of an isolated Bsp Man4 polypeptide or fragment or variant thereof under conditions suitable for breaking down mannan-containing polymers.

[0198] The Bsp Man4 polypeptides of the present disclosure are useful as additives to feed for non-human animals. The term non-human animal includes all non-ruminant and ruminant animals. In a particular embodiment, the non-ruminant animal, is selected from the group consisting of, but not limited to, horses and monogastric animals such as, but not limited to, pigs, poultry, swine and fish. In further embodiments, the pig may be, but not limited to, a piglet, a growing pig, and a sow; the poultry may be, but not limited to, a turkey, a duck and a chicken including, but not limited to, a broiler chick, a layer; and fish including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans including but not limited to shrimps and prawns.such as poultry and swine, In a further embodiment, the non-human animal is a ruminant animal including, but not limited to, cattle, young calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn, and nilgai. The Bag Man1 polypeptides of the present disclosure are also useful as additives. The Bsp Man4 polypeptides of the present disclosure are also useful for human food. In some embodiments, the Bsp Man4 polypeptides are used to pretreat the feed instead of as a feed additive. In some preferred embodiment, the Bsp Man4 polypeptides are added to or used to pretreat feed for weanling pigs, nursery pigs, piglets, fattening pigs, growing pigs, finishing pigs, laying hens, broiler chicks, turkeys. In some embodiment, the Bsp Man4 polypeptides are added to or used to pretreat feed from plant material such as palm kernel, coconut, konjac, locust bean gum, gum guar, soy beans, barley, oats, flax, wheat, corn, linseed, citrus pulp, cottonseed, groundnut, rapeseed, sunflower, peas, and lupines.

[0199] Since the Bsp Man4 polypeptides of the present disclosure are thermostable enzymes, they find used in processes of producing pelleted feed in which heat is applied to the feed mixture before the pelleting step, as it is the case in most commercial pellet mills. The Bsp Man4 polypeptides are added to the other feed ingredients in advance of the pelleting step or after the pelleting step to the already formed feed pellets.

[0200] In compositions containing any of the disclosed Bsp Man4 polypeptides intended for food processing or as a feed supplement, the compositions optionally contain other substituents such as coloring agents, aroma compounds, stabilizers, vitamins, minerals, other feed or food enhancing enzymes and the like. This applies in particular to the so-called pre-mixes. Food additives according to this present invention may be combined with other food components to produce processed food products. The resulting, combined food additive is mixed in an appropriate amount with other food components such as cereal or plant proteins to form a processed food product.

[0201] Accordingly, the present invention relates to an animal feed composition and/or animal feed additive composition and/or pet food comprising the Bsp Man4 polypeptides.

[0202] The present invention further relates to a method for preparing such animal feed composition and/or animal feed additive composition and/or pet food comprising mixing the Bsp Man4 polypeptides with one or more animal feed ingredients and/or animal feed additive ingredients and/or pet food ingredients.

[0203] Furthermore, the present invention relates to the use of the Bsp Man4 polypeptides in the preparation of an animal feed composition and/or animal feed additive composition and/or pet food.

[0204] In the present context, it is intended that the term pet food is understood to mean a food for a household animal such as, but not limited to dogs, cats, gerbils, hamsters, chinchillas, fancy rats, guinea pigs; avian pets, such as canaries, parakeets, and parrots; reptile pets, such as turtles, lizards and snakes; and aquatic pets, such as tropical fish and frogs.

[0205] The terms animal feed composition, feedstuff and fodder are used interchangeably and may comprise one or more feed materials selected from the group comprising a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as corn gluten meal, Distillers Dried Grain Solubles (DDGS) (particularly corn based Distillers Dried Grain Solubles (cDDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from vegetable and animal sources; e) minerals and vitamins.

VIIIa. Bsp Man4 Polypeptides for Fermented Beverages, Such as Beer

[0206] In aspects of the invention the food composition or additive may be liquid or solid.

[0207] In an aspect of the invention the food composition is a beverage, including, but not limited to, a fermented beverage such as beer and wine, comprising any of the Bsp Man4 polypeptides of the invention.

[0208] In the context of the present invention, the term "fermented beverage" is meant to comprise any beverage produced by a method comprising a fermentation process, such as a microbial fermentation, such as a bacterial and/or yeast fermentation.

[0209] In an aspect of the invention the fermented beverage is beer. The term "beer" is meant to comprise any fermented wort produced by fermentation/brewing of a starch-containing plant material. Often, beer is produced from malt or adjunct, or any combination of malt and adjunct as the starch-containing plant material. As used herein the term "malt" is understood as any malted cereal grain, such as malted barley or wheat.

[0210] As used herein the term "adjunct" refers to any starch and/or sugar containing plant material which is not malt, such as barley or wheat malt. As examples of adjuncts, mention can be made of materials such as common corn grits, refined corn grits, brewer's milled yeast, rice, sorghum, refined corn starch, barley, barley starch, dehusked barley, wheat, wheat starch, torrified cereal, cereal flakes, rye, oats, potato, tapioca, cassaya and syrups, such as corn syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheat syrups, and the like may be used as a source of starch

[0211] As used herein, the term "mash" refers to an aqueous slurry of any starch and/or sugar containing plant material such as grist, e.g. comprising crushed barley malt, crushed barley, and/or other adjunct or a combination hereof, mixed with water later to be separated into wort and spent grains.

[0212] As used herein, the term "wort" refers to the unfermented liquor run-off following extracting the grist during mashing.

[0213] In another aspect the invention relates to a method of preparing a fermented beverage such as beer comprising mixing any of the Bsp Man4 polypeptides of the invention with malt or adjunct.

[0214] Examples of beers comprise: full malted beer, beer brewed under the "Reinheitsgebot", ale, IPA, lager, bitter, Happoshu (second beer), third beer, dry beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock beer, stout, malt liquor, non-alcoholic beer, non-alcoholic malt liquor and the like, but also alternative cereal and malt beverages such as fruit flavoured malt beverages, e.g. citrus flavoured, such as lemon-, orange-, lime-, or berry-flavoured malt beverages, liquor flavoured malt beverages, e.g., vodka-, rum-, or tequila-flavoured malt liquor, or coffee flavoured malt beverages, such as caffeine-flavoured malt liquor, and the like.

[0215] One aspect of the invention relates to the use of any of the Bsp Man4 polypeptides according to the invention in the production of a fermented beverage, such as a beer.

[0216] Another aspect concerns a method of providing a fermented beverage comprising the step of contacting a mash and/or a wort with any of the Bsp Man4 polypeptides of the current invention.

[0217] A further aspect relates to a method of providing a fermented beverage comprising the steps of: (a) preparing a mash, (b) filtering the mash to obtain a wort, and (c) fermenting the wort to obtain a fermented beverage, such as a beer, wherein any of the Bsp Man4 polypeptides is added to: (i) the mash of step (a) and/or (ii) the wort of step (b) and/or (iii) the wort of step (c).

[0218] According to yet another aspect, a fermented beverage, such as a beer, is produced or provided by a method comprising the step(s) of (1) contacting a mash and/or a wort with any of the Bsp Man4 polypeptides of the current invention; and/or (2) (a) preparing a mash, (b) filtering the mash to obtain a wort, and (c) fermenting the wort to obtain a fermented beverage, such as a beer, wherein any of the Bsp Man4 polypeptides is added to: (i) the mash of step (a) and/or (ii) the wort of step (b) and/or (iii) the wort of step (c).

[0219] Particular embodiments pertains to any of the above use, method or fermented beverage, wherein said fermented beverage is a beer, such as full malted beer, beer brewed under the "Reinheitsgebot", ale, IPA, lager, bitter, Happoshu (second beer), third beer, dry beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock beer, stout, malt liquor, non-alcoholic beer, non-alcoholic malt liquor and the like, but also alternative cereal and malt beverages such as fruit flavoured malt beverages, e.g., citrus flavoured, such as lemon-, orange-, lime-, or berry-flavoured malt beverages, liquor flavoured malt beverages, e.g., vodka-, rum-, or tequila-flavoured malt liquor, or coffee flavoured malt beverages, such as caffeine-flavoured malt liquor, and the like.

VIII. Bsp Man4 Polypeptides for Treating Coffee Extracts

[0220] The Bsp Man4 polypeptides described herein may also be used for hydrolyzing galactomannans present in liquid coffee extracts. In certain preferred embodiments, the Bsp Man4 polypeptides are used to inhibit gel formation during freeze drying of liquid coffee extracts. The decreased viscosity of the extract reduces the energy consumption during drying. In certain other preferred embodiments, the Bsp Man4 polypeptides are applied in an immobilized form in order to reduce enzyme consumption and avoid contamination of the coffee extract This use is further disclosed in EP 676 145.

[0221] In general terms the coffee extract is incubated in the presence of an isolated Bsp Man4 polypeptide or fragment or variant thereof under conditions suitable for hydrolyzing galactomannans present in liquid coffee extract.

VIIIc Bsp Man4 Polypeptides for Use in Bakery Food Products

[0222] In another aspect the invention relates to a method of preparing baked products comprising addition of any of the Bsp Man4 polypeptides of the invention to dough, followed by baking the dough. Examples of baked products are well known to those skilled in the art and include breads, rolls, puff pastries, sweet fermented doughs, buns, cakes, crackers, cookies, biscuits, waffles, wafers, tortillas, breakfast cereals, extruded products, and the like.

[0223] Any of the Bsp Man4 polypeptides of the invention may be added to dough as part of a bread improver composition. Bread improvers are compositions containing a variety of ingredients, which improve dough properties and the quality of bakery products, e.g. bread and cakes. Bread improvers are often added in industrial bakery processes because of their beneficial effects e.g. the dough stability and the bread texture and volume. Bread improvers usually contain fats and oils as well as additives like emulsifiers, enzymes, antioxidants, oxidants, stabilizers and reducing agents. In addition to any of the Bsp Man4 polypeptides of the present invention, other enzymes which may also be present in the bread improver or which may be otherwise used in conjunction with any of the Bsp Man4 polypeptides of the present invention include amylases, hemicellulases, amylolytic complexes, lipases, proteases, xylanases, pectinases, pullulanases, non starch polysaccharide degrading enzymes and redox enzymes like glucose oxidase, lipoxygenase or ascorbic acid oxidase.

[0224] In a preferred bakery aspect of the current invention, any of the Bsp Man4 polypeptides of the invention may be added to dough as part of a bread improver composition which also comprises a glucomannan and/or galactomannan source such as konjac gum, guar gum, locust bean gum (Ceratonia siliqua), copra meal, ivory nut mannan (Phyteleohas macrocarpa), seaweed mannan extract, coconut meal, and the cell wall of brewers yeast (may be dried, or used in the form of brewers yeast extract). Other acceptable mannan derivatives for use in the current invention include unbranched .beta.-1,4-linked mannan homopolymer and manno-oligosaccharides (mannobiose, mannotriose, mannotetraose and mannopentoase). The combination of any of the Bsp Man4 polypeptides of the invention with a glucomannan and/or galactomannan and/or galatoglucomannan further improves the dough tolerance, dough flexibility and dough stickiness, improves the bread crumb structure and retards staling of the bread, and the mannanase hydrolysates act as soluble prebiotics by promoting the growth of lactic acid bacteria commonly associated with good health when found at favourable population densities in the colon.

[0225] A further aspect of the invention relates to the use of any of the Bsp Man4 polypeptides of the invention in dough to improve dough tolerance, flexibility and stickiness. Preferably the dough to which any of the Bsp Man4 polypeptides of the invention may be added is not a pure white flour dough, but comprises bran or oat, rice, millet, maize, or legume flour in addition to or instead of pure wheat flour.

[0226] A yet further aspect of the invention relates to the use of any of the Bsp Man4 polypeptides of the invention in dough to improve the crumb structure and retard staling in the final baked product, such as bread.

VIIIc Bsp Man4 Polypeptides for Use in Dairy Food Products

[0227] In one aspect of the current invention, any of the Bsp Man4 polypeptides of the invention may be added to milk or any other dairy product to which has also been added a glucomannan and/or galactomannan. Typical glucomannan and/or galactomannan sources are listed above in the bakery aspects, and include guar or konjac gum. The combination of any of the Bsp Man4 polypeptides of the invention with a glucomannan and/or galactomannan releases mannanase hydrolysates (mannooligosaccharides) which act as soluble prebiotics by promoting the selective growth and proliferation of probiotic bacteria (especially Bifidobacteria and Lactobacillus lactic acid bacteria) commonly associated with good health when found at favourable population densities in the large intestine or colon.

[0228] In another aspect the invention relates to a method of preparing milk or dairy products comprising addition of any of the Bsp Man4 polypeptides of the invention and addition of any glucomannan or galactomannan or galactoglucomannan.

[0229] In another aspect of the invention any of the Bsp Man4 polypeptides of the invention are used in combination with any glucomannan or galactomannan prior to or following addition to a dairy based foodstuff to produce a dairy based foodstuff comprising prebiotic mannan hydrolysates. In a further aspect of the invention the thus produced mannooligosacharide-containing dairy product is capable of increasing the population of beneficial human intestinal microflora, and in a yet further aspect of the current invention the dairy based foodstuff may comprise any of the Bsp Man4 polypeptides of the current invention together with any source of glucomannan and/or galactomannan and/or galactoglucomannan, and a dose sufficient for inoculation of at least one strain of bacteria (such as Bifidobacteria or Lactobacillus) known to be of benefit in the human large intestine. Preferably said dairy-based foodstuff is a yoghurt or milk drink.

IX. Bsp Man4 Polypeptides for Paper Pulp Bleaching

[0230] The Bsp Man4 polypeptides described herein find further use in the enzyme aided bleaching of paper pulps such as chemical pulps, semi-chemical pulps, kraft pulps, mechanical pulps or pulps prepared by the sulfite method. In general terms, paper pulps are incubated with an isolated Bsp Man4 polypeptide or fragment or variant thereof under conditions suitable for bleaching the paper pulp.

[0231] In some embodiments, the pulps are chlorine free pulps bleached with oxygen, ozone, peroxide or peroxyacids. In some embodiments, the Bsp Man4 polypeptides are used in enzyme aided bleaching of pulps produced by modified or continuous pulping methods that exhibit low lignin contents. In some other embodiments, the Bsp Man4 polypeptides are applied alone or preferably in combination with xylanase and/or endoglucanase and/or alpha-galactosidase and/or cellobiohydrolase enzymes.

X. Bsp Man4 Polypeptides for Degrading Thickeners

[0232] Galactomannans such as guar gum and locust bean gum are widely used as thickening agents e.g., in food and print paste for textile printing such as prints on T-shirts. Thus the Bsp Man4 polypeptides described herein also find use in reducing the thickness or viscosity of mannan-containing substrates. In certain embodiments, the Bsp Man4 polypeptides described herein are used for reducing the viscosity of residual food in processing equipment and thereby facilitate cleaning after processing. In certain other embodiments, the disclosed Bsp Man4 polypeptides are used for reducing viscosity of print paste, thereby facilitating wash out of surplus print paste after textile printings. In general terms, a mannan-containing substrate is incubated with an isolated Bsp Man4 polypeptide or fragment or variant thereof under conditions suitable for reducing the viscosity of the mannan-containing substrate.

[0233] Other aspects and embodiments of the present compositions and methods will be apparent from the foregoing description and following examples.

EXAMPLES

[0234] The following examples are provided to demonstrate and illustrate certain preferred embodiments and aspects of the present disclosure and should not be construed as limiting.

[0235] In the experimental disclosure which follows, the following abbreviations apply: M (molar); mM (millimolar); .mu.M (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); .mu.mol (micromoles); nmol (nanomoles); g and gm (grams); mg (milligrams); .mu.g (micrograms); pg (picograms); L (liters); ml and mL (milliliters); .mu.l and .mu.L (microliters); cm (centimeters); mm (millimeters); .mu.m (micrometers); nm (nanometers); U (units); MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s) (hour/hours); .degree. C. (degrees Centigrade); QS (quantity sufficient); ND (not done); rpm (revolutions per minute); H.sub.2O (water); dH.sub.2O (deionized water); HCl (hydrochloric acid); aa (amino acid); by (base pair); kb (kilobase pair); kD (kilodaltons); MgCl.sub.2 (magnesium chloride); NaCl (sodium chloride); Ca (calcium); Mg (magnesium); HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); CHES (N-cyclohexyl-2-aminoethanesulfonic acid); w/v (weight to volume); v/v (volume to volume); g (gravity); OD (optical density); ppm (parts per million); m-(meta-); o-(ortho-); p-(para-); PAHBAH (p-hydroxybenzoic acid hydrazide); Bsp Man4 (Bacillus sp. mannanase4); SRI (stain removal index); and % SR (percentage stain removal).

Example 1

Cloning of the Bacillus sp. SWT81 Glycosyl Hydrolase Bsp Man4

[0236] Bacillus sp. SWT81 was selected as a potential source for various glycosyl hydrolases and other enzymes, useful for industrial applications. Genomic DNA for sequencing was obtained by first growing Bacillus sp. SWT81 on GAM agar plates (Jones et al., IJSEM, 55:1711-1714, 2005) at 37.degree. C. for 24 h. Cell material was scraped from the plates and used to prepare genomic DNA using the ZF Fungal/Bacterial DNA miniprep kit from Zymo (Cat No. D6005). The genomic DNA was used for genome sequencing and to amplify the Bsp Man4 gene for expression cloning. The entire genome of the Bacillus sp. SWT81 strain was sequenced using Illumina.RTM. sequencing by synthesis (SBS) technology (www.baseclear.com/sequencing/illumina-sequencing/). Genome sequencing and assembly of the sequence data was performed by BaseClear (Leiden, The Netherlands). Contigs were annotated by BioXpr (Namur, Belgium). One of the genes identified this way in Bacillus sp. SWT81 encodes a glycosyl hydrolase with homology to mannanases of various other bacteria as determined from a BLAST search (Altschul et al., J Mol Biol, 215: 403-410, 1990). The sequence of this gene, called the Bsp Man4 gene, is depicted as SEQ ID NO.1. The protein encoded by the Bsp Man4 gene is depicted as SEQ ID NO. 2. At the N-terminus, Bsp Man4 has a 29 amino acid signal peptide as predicted by SignalP-3.0 program (www.cbs.dtu/services/SignalP) set to SignalP-NN system (Emanuelsson et al., Nature Protocols, 2:953-971, 2007). This indicates that Bsp Man4 is a secreted glycosyl hydrolase.

[0237] The nucleotide sequence of the Bsp Man4 coding region is set forth as SEQ ID NO:1. The coding region of the predicted signal peptide sequence is shown in italics.

TABLE-US-00003 atgggaacatggaaaaaggggtttgtgttatttattgtcctaatgttagtttttgatgtatcgatgttgggtg- taaatgtaagcgcttcac aagaagggcgtcaacttaacatggcagatgaggatgcttcaaagtatacgaaggagttatttgcttttcttcaa- gatgtaagtggttcaca agtgttatttggacaacagcatgcaacagatgaaggattaactttaacaaatccagctccaagaacaggttcca- ctcaatctgaagttttc aatgcagttggggattatccagctgtgtttggatgggacacgaatagcctagatggtcgtgaaaagcctggcat- tgcaggtaatgtagaac aaagtataaaaaatacggctcagtccatgaaagtggctcatgatttaggagggattattacactaagcatgcac- ccagataattttgtaac agggggtccttatggtgatacaacagggaatgttgtaaaagaaattcttccaggtggatcaaaacatgcagagt- ttaacgcgtggttggac aatattgctgcgcttgctcacgagctgaaagatgagaatggtgaacctattccgatgatttttcggccattcca- tgaacaaacaggatctt ggttttggtggggagcaagcacaacttcacccgaacaatataaagcgatttttcgttatacagtagaatatttg- cgagatgttaaaggcgt aaataatattttatatggcttttcacctggggcgggacctgctggagatgtaaatcgctatttagaaacatatc- caggggatgattacgtt gatattttcggtattgacaattatgacaataaagacaatgcagggtcagaagcttggttaagtggtatggtcaa- agacttggcgatgatta gccgattagctgaacaaaaagaaaaagtagcggcttttactgagtatgggtacagtgcaaccggaattaatcgt- caagggaatacattaga ctggtacacacgtgtattagatgcgattgctgctgatgaagacgcacgtaaaatatcatacatgttgacatggg- cgaactttggttggccg aataatatgtatgttccttatcgtgatatccacaatgaattaggtggagaccatgagttattaccggactttga- agctttccatgcggatg actacacagcatttcgagatgagataaaaggaaagatatataatactggaaaggaatataccgtttctcctcat- gagccgtttatgtatgt tatatctccgattacaggttctacagtgacaagcgaaacggtaacaatccaagcaaaagtagcgaatgacgaac- acgcaagagtcactttc agggtcgatggttctagtttggaagaagaaatggttttcaatgatgacactttatattatacaggttcttttac- accagatgcagcagtga atggcggagctgttgatgtgattgtagcttattattctagtggagaaaaagtccaagaagaaacaattcgttta- tttgtaaaaattcctga aatgtctttgttaacattaacgtttgatgatgatataaacggaatcaaaagcaatggaacatggcctgaagatg- gtgtaacatctgaaatt gaccacgctattgtagatggagacggcaagttgatgttctctgttcaaggaatgtcacctactgaaacatggca- agagctcaagttagaat taacagaactatcagatgtgaacattgatgcggttaagaaaatgaagtttgacgcgcttatcccagcaggtagt- gaagaaggttcagtcca aggaatcgtacaacttccaccggattgggagacgaaatatgggatgaatgaaacaacgaagtcaataaaagact- tagagactgttactgtt aatggaagcgattataaacggttggaagtgactgtttctatcgacaatcaaggaggagctacaggaatcgcttt- atcattagtaggatccc aactcgatttgttagaacctgtctacatcgataatattgaacttctaaattcctttgaagcaccaccagcagat- tcttttcttgttgatga ttttgaaggttattttggggatgacacgttgttacatcgcaattattctagcaatggagatccaattacactat- cgttaacaagtgagttt aaaaataatggagaatttggattgaagtatgattattcgattggctcgatgggttatgcagggaggcaaacatc- actaggacctgtcgatt ggagcggagctaatgcttttgaattttggatgaaacatggacaacttgaagggaatcatttaactgtacaaatt- cgaataggtgatgttag ctttgaaaaaaatcttgaattaatggatgctcatgaaggtgtagtgacaatcccgttttctgaatttgctccag- ctgcttgggaaaataag cctggcgttatcattgacgaacaaaaattgaaaagagtgagtcaatttgctctttacacaggcggggctagaca- atctggaacaatctact ttgatgatttacgagcggtatatgatgaaagtttaccatcagttccagttccgaaagaggaggaagaggaaaaa- gaggtcgctcctattat ttatcattttgaatctggaattgataattgggaagggggacaagcaacacatagcaatgggcacctcaaagtaa- cggttcgtttaggtgaa ggtcagcaaaccgaagtgaagaaaacatcaaattataatttaacagggtataattatatagtagctaatataaa- acatgacgatacaggaa tgtttggtagtgacccgcttcaagtgaaaatctttacgaaagcaggaggttgggtatgggctgattcaggaaat- caaccgatttactccga cgattatactcaagttgtgtatgatattactactttagctaacaaaaatgcagtccaagaaatcgggtttgaat- ttttggctccttcaggt tcttcagggacgacgaatcctttcatagattcagtagcgattgttacgagtctcgatcaattgtctgagcagcc- agagcagccagaacaac caggaacaccagatactgatgataataaagaggataaagatagaagaaatgtagaagtgaacgaggaaggacaa- aaactacccaaaacagc aacgtcaatatttaattatttgctaattggttttgtttttgtagggattggatttagtctatttatttataaaa- gaagaaaaacagtg.

[0238] The amino acid sequence of the protein encoded by the Bsp Man 4 gene is set forth as SEQ ID NO:2. The predicted signal peptide is shown in italics.

TABLE-US-00004 MGTWKKGFVLFIVLMLVFDVSMLGVNVSASQEGRQLNMADEDASKYTKEL FAFLQDVSGSQVLFGQQHATDEGLTLTNPAPRTGSTQSEVFNAVGDYPAV FGWDTNSLDGREKPGIAGNVEQSIKNTAQSMKVAHDLGGIITLSMHPDNF VTGGPYGDTTGNVVKEILPGGSKHAEFNAWLDNIAALAHELKDENGEPIP MIFRPFHEQTGSWFWWGASTTSPEQYKAIFRYTVEYLRDVKGVNNILYGF SPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDNKDNAGSEAWLSGMVKDL AMISRLAEQKEKVAAFTEYGYSATGINRQGNTLDWYTRVLDAIAADEDAR KISYMLTWANFGWPNNMYVPYRDIHNELGGDHELLPDFEAFHADDYTAFR DEIKGKIYNTGKEYTVSPHEPFMYVISPITGSTVTSETVTIQAKVANDEH ARVTFRVDGSSLEEEMVFNDDTLYYTGSFTPDAAVNGGAVDVIVAYYSSG EKVQEETIRLFVKIPEMSLLTLTFDDDINGIKSNGTWPEDGVTSEIDHAI VDGDGKLMFSVQGMSPTETWQELKLELTELSDVNIDAVKKMKFDALIPAG SEEGSVQGIVQLPPDWETKYGMNETTKSIKDLETVTVNGSDYKRLEVTVS IDNQGGATGIALSLVGSQLDLLEPVYIDNIELLNSFEAPPADSFLVDDFE GYFGDDTLLHRNYSSNGDPITLSLTSEFKNNGEFGLKYDYSIGSMGYAGR QTSLGPVDWSGANAFEFWMKHGQLEGNHLTVQIRIGDVSFEKNLELMDAH EGVVTIPFSEFAPAAWENKPGVIIDEQKLKRVSQFALYTGGARQSGTIYF DDLRAVYDESLPSVPVPKEEEEEKEVAPIIYHFESGIDNWEGGQATHSNG HLKVTVRLGEGQQTEVKKTSNYNLTGYNYIVANIKHDDTGMFGSDPLQVK IFTKAGGWVWADSGNQPIYSDDYTQVVYDITTLANKNAVQEIGFEFLAPS GSSGTTNPFIDSVAIVTSLDQLSEQPEQPEQPGTPDTDDNKEDKDRRNVE VNEEGQKLPKTATSIFNYLLIGFVFVGIGFSLFIYKRRKTV.

Example 2

Expression of Bacillus sp. Glycosyl Hydrolase (Bsp Man4)

[0239] The Bsp Man4 gene was amplified from genomic DNA of Bacillus sp. using the following primers: Primer 1(BssHII) 5'-TGAGCGCGCA GGCAGCTGGT AAATCACAAG AAGGGCGTCA ACT-3' (SEQ ID NO:3), and Primer 2 (XhoI) 5'-CGCCTCGAGT TACACTGTTT TTCTTCTTTT AT-3' (SEQ ID NO:4). After digestion with BssHII/XhoI, the PCR product was cloned into the p2JM103BBI expression vector (Vogtentanz, Protein Expr Purif, 55:40-52, 2007) digested with the same restriction enzymes. Ligation of this DNA fragment to the PCR amplified gene encoding the Bsp Man4 mature protein resulted in the addition of three codons from the 3' end of the nucleic acid encoding the Bacillus subtilis AprE pro-peptide to the 5' end of the coding region of the mature form of Bsp Man4. The resulting plasmid was labeled pZQ186 (aprE-Bsp Man4). A plasmid map of pZQ186 is provided as FIG. 1. Following the natural signal peptidase cleavage in the host, the recombinant Bsp Man4 protein produced in this manner has three additional amino acids (Ala-Gly-Lys) at its amino-terminus. The sequence of Bsp Man4 gene was confirmed by DNA sequencing (SEQ ID NO:5).

[0240] The Bsp Man4 protein was produced in Bacillus subtilis cells using previously described methods (Vogtentanz, Protein Expr Purif, 55:40-52, 2007). The protein was secreted into the extracellular medium and filtered culture medium was used to perform the cleaning assay and the pH and temperature profile experiments. The dosing was based on total protein determined by a Bradford type assay using the Biorad protein assay (500-0006EDU) and corrected for purity as determined by SDS-PAGE using a Criterion stain free system from Bio-Rad).

[0241] Analysis of the culture supernatant on an SDS-PAGE gel revealed three separate and distinct protein bands falling within the expected molecular weight range of Bsp Man4. The concentrated culture supernatant of Bacillus cells expressing Bsp Man4 was used for purification using three chromatography columns. Concentrated culture supernatant buffered in 20 mM Tris pH 7.5 was loaded on an anion exchange Sepharose column (Sepharose-Q FF, XK 16/10) equilibrated with 20 mM Tris pH 7.5. The protein was eluted from the column using a linear gradient of equilibration/wash buffer to 20 mM Tris, pH 7.5 buffer containing 0.5 M NaCl. The pooled sample was adjusted to final concentration of 1M (NH.sub.4).sub.2SO.sub.4 and loaded onto a hydrophobic interaction chromatography column (HiLoad Phenyl HP, 16/10) equilibrated with 20 mM sodium phosphate pH 6.0, 1M (NH.sub.4).sub.2SO.sub.4 buffer. The protein was eluted from the column using a linear gradient of equilibration/wash buffer to 20 mM sodium phosphate pH 6.0. Three fractions, approximately 100 kD (alpha), 70 kD (beta), and 50 kD (gamma), all with mannanase activity, were collected separately. Each fraction was loaded onto the gel filtration HiLoad Superdex 75 pg 26/60 column (for 50 kD and 70 kD fractions) or HiLoad Superdex 200 pg 26/60 column (for 100 kD fraction), and the mobile phase used was 20 mM sodium phosphate, pH 7.0, containing 0.15 M NaCl. The pooled samples from gel filtration columns were concentrated to obtain purified protein samples.

[0242] Nucleotide sequence of Bsp Man4 gene from expression plasmid pZQ186 is set forth as SEQ ID NO:5. The aprE signal sequence is shown in italics.

TABLE-US-00005 gtgagaagcaaaaaattgtggatcagcttgttgtttgcgttaacgttaatctttacgatggcgttcagcaaca- tgagcgcgcaggcagct ggtaaatcacaagaagggcgtcaacttaacatggcagatgaggatgcttcaaagtatacgaaggagttatttgc- ttttcttcaagatgta agtggttcacaagtgttatttggacaacagcatgcaacagatgaaggattaactttaacaaatccagctccaag- aacaggttccactcaa tctgaagttttcaatgcagttggggattatccagctgtgtttggatgggacacgaatagcctagatggtcgtga- aaagcctggcattgca ggtaatgtagaacaaagtataaaaaatacggctcagtccatgaaagtggctcatgatttaggagggattattac- actaagcatgcaccca gataattttgtaacagggggtccttatggtgatacaacagggaatgttgtaaaagaaattcttccaggtggatc- aaaacatgcagagttt aacgcgtggttggacaatattgctgcgcttgctcacgagctgaaagatgagaatggtgaacctattccgatgat- ttttcggccattccat gaacaaacaggatcttggttttggtggggagcaagcacaacttcacccgaacaatataaagcgatttttcgtta- tacagtagaatatttg cgagatgttaaaggcgtaaataatattttatatggcttttcacctggggcgggacctgctggagatgtaaatcg- ctatttagaaacatat ccaggggatgattacgttgatattttcggtattgacaattatgacaataaagacaatgcagggtcagaagcttg- gttaagtggtatggtc aaagacttggcgatgattagccgattagctgaacaaaaagaaaaagtagcggcttttactgagtatgggtacag- tgcaaccggaattaat cgtcaagggaatacattagactggtacacacgtgtattagatgcgattgctgctgatgaagacgcacgtaaaat- atcatacatgttgaca tgggcgaactttggttggccgaataatatgtatgttccttatcgtgatatccacaatgaattaggtggagacca- tgagttattaccggac tttgaagctttccatgcggatgactacacagcatttcgagatgagataaaaggaaagatatataatactggaaa- ggaatataccgtttct cctcatgagccgtttatgtatgttatatctccgattacaggttctacagtgacaagcgaaacggtaacaatcca- agcaaaagtagcgaat gacgaacacgcaagagtcactttcagggtcgatggttctagtttggaagaagaaatggttttcaatgatgacac- tttatattatacaggt tcttttacaccagatgcagcagtgaatggcggagctgttgatgtgattgtagcttattattctagtggagaaaa- agtccaagaagaaaca attcgtttatttgtaaaaattcctgaaatgtctttgttaacattaacgtttgatgatgatataaacggaatcaa- aagcaatggaacatgg cctgaagatggtgtaacatctgaaattgaccacgctattgtagatggagacggcaagttgatgttctctgttca- aggaatgtcacctact gaaacatggcaagagctcaagttagaattaacagaactatcagatgtgaacattgatgcggttaagaaaatgaa- gtttgacgcgcttatc ccagcaggtagtgaagaaggttcagtccaaggaatcgtacaacttccaccggattgggagacgaaatatgggat- gaatgaaacaacgaag tcaataaaagacttagagactgttactgttaatggaagcgattataaacggttggaagtgactgtttctatcga- caatcaaggaggagct acaggaatcgctttatcattagtaggatcccaactcgatttgttagaacctgtctacatcgataatattgaact- tctaaattcctttgaa gcaccaccagcagattcttttcttgttgatgattttgaaggttattttggggatgacacgttgttacatcgcaa- ttattctagcaatgga gatccaattacactatcgttaacaagtgagtttaaaaataatggagaatttggattgaagtatgattattcgat- tggctcgatgggttat gcagggaggcaaacatcactaggacctgtcgattggagcggagctaatgcttttgaattttggatgaaacatgg- acaacttgaagggaat catttaactgtacaaattcgaataggtgatgttagctttgaaaaaaatcttgaattaatggatgctcatgaagg- tgtagtgacaatcccg ttttctgaatttgctccagctgcttgggaaaataagcctggcgttatcattgacgaacaaaaattgaaaagagt- gagtcaatttgctctt tacacaggcggggctagacaatctggaacaatctactttgatgatttacgagcggtatatgatgaaagtttacc- atcagttccagttccg aaagaggaggaagaggaaaaagaggtcgctcctattatttatcattttgaatctggaattgataattgggaagg- gggacaagcaacacat agcaatgggcacctcaaagtaacggttcgtttaggtgaaggtcagcaaaccgaagtgaagaaaacatcaaatta- taatttaacagggtat aattatatagtagctaatataaaacatgacgatacaggaatgtttggtagtgacccgcttcaagtgaaaatctt- tacgaaagcaggaggt tgggtatgggctgattcaggaaatcaaccgatttactccgacgattatactcaagttgtgtatgatattactac- tttagctaacaaaaat gcagtccaagaaatcgggtttgaatttttggctccttcaggttcttcagggacgacgaatcctttcatagattc- agtagcgattgttacg agtctcgatcaattgtctgagcagccagagcagccagaacaaccaggaacaccagatactgatgataataaaga- ggataaagatagaaga aatgtagaagtgaacgaggaaggacaaaaactacccaaaacagcaacgtcaatatttaattatttgctaattgg- ttttgtttttgtaggg attggatttagtctatttatttataaaagaagaaaaacagtg.

[0243] The amino acid sequence of Bsp Man4 expressed from plasmid pZQ186 is set forth as SEQ ID NO:6. The signal sequence is shown in italics, while the three amino acid amino-terminal extension is shown in bold.

TABLE-US-00006 MRSKKLWISLLFALTLIFTMAFSNMSAQAAGKSQEGRQLNMADEDASKYT KELFAFLQDVSGSQVLFGQQHATDEGLTLTNPAPRTGSTQSEVFNAVGDY PAVFGWDTNSLDGREKPGIAGNVEQSIKNTAQSMKVAHDLGGIITLSMHP DNFVTGGPYGDTTGNVVKEILPGGSKHAEFNAWLDNIAALAHELKDENGE PIPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRYTVEYLRDVKGVNNIL YGFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDNKDNAGSEAWLSGMV KDLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTLDWYTRVLDAIAADE DARKISYMLTWANFGWPNNMYVPYRDIHNELGGDHELLPDFEAFHADDYT AFRDEIKGKIYNTGKEYTVSPHEPFMYVISPITGSTVTSETVTIQAKVAN DEHARVTFRVDGSSLEEEMVFNDDTLYYTGSFTPDAAVNGGAVDVIVAYY SSGEKVQEETIRLFVKIPEMSLLTLTFDDDINGIKSNGTWPEDGVTSEID HAIVDGDGKLMFSVQGMSPTETWQELKLELTELSDVNIDAVKKMKFDALI PAGSEEGSVQGIVQLPPDWETKYGMNETTKSIKDLETVTVNGSDYKRLEV TVSIDNQGGATGIALSLVGSQLDLLEPVYIDNIELLNSFEAPPADSFLVD DFEGYFGDDTLLHRNYSSNGDPITLSLTSEFKNNGEFGLKYDYSIGSMGY AGRQTSLGPVDWSGANAFEFWMKHGQLEGNHLTVQIRIGDVSFEKNLELM DAHEGVVTIPFSEFAPAAWENKPGVIIDEQKLKRVSQFALYTGGARQSGT IYFDDLRAVYDESLPSVPVPKEEEEEKEVAPIIYHFESGIDNWEGGQATH SNGHLKVTVRLGEGQQTEVKKTSNYNLTGYNYIVANIKHDDTGMFGSDPL QVKIFTKAGGWVWADSGNQPIYSDDYTQVVYDITTLANKNAVQEIGFEFL APSGSSGTTNPFIDSVAIVTSLDQLSEQPEQPEQPGTPDTDDNKEDKDRR NVEVNEEGQKLPKTATSIFNYLLIGFVFVGIGFSLFIYKRRKTV.

[0244] The amino acid sequence of the mature form of Bsp Man4 is set forth as SEQ ID NO:7. The three amino acid N-terminal extension is shown in bold.

TABLE-US-00007 AGKSQEGRQLNMADEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTL TNPAPRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKN TAQSMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAE FNAWLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQY KAIFRYTVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIF GIDNYDNKDNAGSEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGI NRQGNTLDWYTRVLDAIAADEDARKISYMLTWANFGWPNNMYVPYRDIHN ELGGDHELLPDFEAFHADDYTAFRDEIKGKIYNTGKEYTVSPHEPFMYVI SPITGSTVTSETVTIQAKVANDEHARVTFRVDGSSLEEEMVFNDDTLYYT GSFTPDAAVNGGAVDVIVAYYSSGEKVQEETIRLFVKIPEMSLLTLTFDD DINGIKSNGTWPEDGVTSEIDHAIVDGDGKLMFSVQGMSPTETWQELKLE LTELSDVNIDAVKKMKFDALIPAGSEEGSVQGIVQLPPDWETKYGMNETT KSIKDLETVTVNGSDYKRLEVTVSIDNQGGATGIALSLVGSQLDLLEPVY IDNIELLNSFEAPPADSFLVDDFEGYFGDDTLLHRNYSSNGDPITLSLTS EFKNNGEFGLKYDYSIGSMGYAGRQTSLGPVDWSGANAFEFWMKHGQLEG NHLTVQIRIGDVSFEKNLELMDAHEGVVTIPFSEFAPAAWENKPGVIIDE QKLKRVSQFALYTGGARQSGTIYFDDLRAVYDESLPSVPVPKEEEEEKEV APIIYHFESGIDNWEGGQATHSNGHLKVTVRLGEGQQTEVKKTSNYNLTG YNYIVANIKHDDTGMFGSDPLQVKIFTKAGGWVWADSGNQPIYSDDYTQV VYDITTLANKNAVQEIGFEFLAPSGSSGTTNPFIDSVAIVTSLDQLSEQP EQPEQPGTPDTDDNKEDKDRRNVEVNEEGQKLPKTATSIFNYLLIGFVFV GIGFSLFIYKRRKTV.

[0245] The amino acid sequence of the mature form of Bsp Man4 based on the naturally occurring gene sequence is set forth as SEQ ID NO:8.

TABLE-US-00008 SQEGRQLNMADEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTLTNP APRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKNTAQ SMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAEFNA WLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAI FRYTVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGID NYDNKDNAGSEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQ GNTLDWYTRVLDAIAADEDARKISYMLTWANFGWPNNMYVPYRDIHNELG GDHELLPDFEAFHADDYTAFRDEIKGKIYNTGKEYTVSPHEPFMYVISPI TGSTVTSETVTIQAKVANDEHARVTFRVDGSSLEEEMVFNDDTLYYTGSF TPDAAVNGGAVDVIVAYYSSGEKVQEETIRLFVKIPEMSLLTLTFDDDIN GIKSNGTWPEDGVTSEIDHAIVDGDGKLMFSVQGMSPTETWQELKLELTE LSDVNIDAVKKMKFDALIPAGSEEGSVQGIVQLPPDWETKYGMNETTKSI KDLETVTVNGSDYKRLEVTVSIDNQGGATGIALSLVGSQLDLLEPVYIDN IELLNSFEAPPADSFLVDDFEGYFGDDTLLHRNYSSNGDPITLSLTSEFK NNGEFGLKYDYSIGSMGYAGRQTSLGPVDWSGANAFEFWMKHGQLEGNHL TVQIRIGDVSFEKNLELMDAHEGVVTIPFSEFAPAAWENKPGVIIDEQKL KRVSQFALYTGGARQSGTIYFDDLRAVYDESLPSVPVPKEEEEEKEVAPI IYHFESGIDNWEGGQATHSNGHLKVTVRLGEGQQTEVKKTSNYNLTGYNY IVANIKHDDTGMFGSDPLQVKIFTKAGGWVWADSGNQPIYSDDYTQVVYD ITTLANKNAVQEIGFEFLAPSGSSGTTNPFIDSVAIVTSLDQLSEQPEQP EQPGTPDTDDNKEDKDRRNVEVNEEGQKLPKTATSIFNYLLIGFVFVGIG FSLFIYKRRKTV.

Example 3

pH Profile of Bsp Man4

[0246] The pH profile of Bsp Man4 was determined using the beta-mannazyme tablet assay from Megazyme (Tmnz 1/02; Azurine-crosslinked carob galactomannan) with minor modifications to the suggested protocol. The assay was performed in 50 mm Acetate/Bis-Tris/HEPES/CHES buffer adjusted to pH values between 4 and 11. The enzyme solution was diluted into the assay buffer and 500 .mu.l of the enzyme solution was equilibrated at 40.degree. C. before adding one substrate tablet. After 10 minutes, the reaction was stopped by adding 10 ml 2% Tris pH 12. The tubes were left at room temperature for 5 minutes, stirred and the liquid filtered through a Whatman No.1 paper filter. Release of blue dye from the substrate was quantified by measuring the optical density at 590 nm. Enzyme activity at each pH was reported as relative activity where the activity at the pH optimum was set to 100%. The pH profile of Bsp Man4 is shown in FIG. 2A. Bsp Man 4 was found to have an optimum pH at about 6.5, and was found to retain greater than 70% of maximum activity between pH 6.0 and 8.5.

[0247] The pH profile of Mannastar.TM. was studied by assaying for mannanase activity at varying pH values ranging from 4-11 using the beta-mannazyme tablet assay (Megazyme, Ireland). The generation of water soluble dye fragments was monitored after 10 min at OD 590 nm at each pH value. A pH profile plot was made by setting the highest OD value for activity to 100 and determining the activity at the other pH values relative to the highest OD value. The pH profile of Mannastar.TM. is shown in FIG. 2B. Mannastar.TM. was found to retain greater than 70% of maximum activity between pH 4 and 7.5.

Example 4

Temperature Profile of Bsp Man4

[0248] The temperature optimum of purified Bsp Man4 was determined by assaying for mannanase activity at temperatures varying between 20.degree. C. and 75.degree. C. for 10 minutes in 50 mM sodium citrate buffer at pH 6. The activity was reported as relative activity where the activity at the temperature optimum was set to 100%. The temperature profile of Bsp Man4 is shown in FIG. 3A. Bsp Man 4 was found to have an optimum temperature of about 60.degree. C., and was found to retain greater than 70% of maximum activity between 55.degree. C. and 65.degree. C.

[0249] The temperature profile of Mannastar.TM. was studied by assaying for mannanase activity at varying temperatures ranging from 20.degree. C. to 75.degree. C. using the beta-mannazyme tablet assay (Megazyme, Ireland) in 50 mM sodium acetate buffer at pH 6. The generation of water soluble dye fragments was monitored after 10 min at OD 590 nm at each temperature. The temperature profile was made by setting the highest OD value for activity to 100% and determining the activity at the other temperatures relative to the maximum. The temperature profile of Mannastar.TM. is shown in FIG. 3B. Mannastar.TM. was found to retain greater than 70% maximum activity 55.degree. C. and 75.degree. C.

Example 5

Mannanase Activity of Bsp Man4

[0250] Bsp Man4 (EC number 3.2.1.78) belongs to the CAZy number GH26 glycosyl hydrolase family. Three forms of Bsp Man4 were identified by SDS-PAGE: alpha (MW.about.100 kDa); beta (MW.about.70 kDa); and gamma (MW.about.50 kDa). The sample containing the gamma form of Bsp Man4 was a mixture including unrelated proteins, with Bsp Man4 present at 30% w/w. The beta 1-4 mannanase activity of the three forms of Bsp Man4 was measured using 1% Megazyme Low Viscosity Carob Galactomannan (Megazyme International, Ireland) as a substrate in a PAHBAH assay (Lever, Anal Biochem, 47:248, 1972). The assay was performed either in 50 mM sodium acetate pH 5, 0.005% Tween-80 buffer at 50.degree. C. for 10 minutes or 50 mM HEPES pH 8.2, 0.005% Tween-80 buffer at 30.degree. C. for 30 minutes. A standard curve using mannose was created for each buffer and used to calculate enzyme activity units. Enzyme Specific Activity Unit Definition: One mannanase unit is defined as the amount of enzyme required to generate 1 mole of mannose reducing sugar equivalents per minute under the conditions of the assay. FIG. 4A shows the mannanase activity displayed by the three forms of Bsp Man4 at pH 8.2. FIG. 4B shows the mannanase activity displayed by the three forms of Bsp Man4 at pH 5.0.

Example 6

Cleaning Performance of Bsp Man4 and Fragments Thereof

[0251] The cleaning performance of Bsp Man4 was tested in a Launder-O-meter LP-2 (Atlas Electric Devices Co., Chicago, Ill.) or equivalent using the CS-43 (Guar Gum), CS-73 (Locust Bean Gum), and PCS-43 (pigment stained Guar Gum) swatches purchased from Center for Testmaterials, The Netherlands. The cleaning performance of Bsp Man4 was tested in combination with a protease (PURAFECT.RTM. or PURAFECT.RTM. Prime). Swatches were cut to 3 cm.times.3 cm in size, read on a Konica Minolta CR-400 reflectometer for pre-wash RGB values, and 4 swatches of each stain type (12 g including ballast soil) were added to each test beaker along with 6 stainless steel balls. Water hardness was adjusted to a final concentration of 100 ppm and used to dilute the detergents. The commercially available detergent OMO color powder (Unilever) was heat-inactivated and used at a dose of 5.25 g/L. The commercially available Small and Mighty bio liquid detergent (Unilever) contained no enzymes and was used without heat-inactivation at a dose of 2.33 g/L. Varying doses (0.25, 1 and 2.5 ppm) of Bsp Man4 along with 0.5 ppm of PURAFECT.RTM. Prime for liquid detergent or 0.8 ppm of PURAFECT.RTM. for powder detergent were added to each beaker. The washing cycle time was 45 minutes at 40.degree. C. After the wash, the swatches were removed, rinsed for 5 minutes in cold tap water, spun in a laundry centrifuge and laid flat in heating cabinet to dry. The dry swatches were covered with dark cloth at room temperature and stain removal was assessed by measuring the RGB values with a Konica Minolta CR-400 reflectometer. Stain removal was calculated using the RGB color values as the difference of the post- and pre-cleaning RGB color measurements for each swatch. The % SR readings for 1 ppm Bsp Man4 dose are shown in FIGS. 5A and 5B.

[0252] Three forms of Bsp Man4 were identified by SDS-PAGE: alpha (MW.about.100 kDa); beta (MW.about.70 kDa); and gamma (MW.about.50 kDa). The sample containing the gamma form of Bsp Man4 was a mixture including unrelated proteins, with Bsp Man4 present at 30% w/w. The cleaning performance of the three forms of Bsp Man4 was tested in a Launder-O-meter LP-2 (Atlas Electric Devices Co., Chicago, Ill.) or equivalent using the CS-43 (Guar Gum) and CS-73 (Locust Bean Gum) swatches purchased from Center for Testmaterials, The Netherlands. The cleaning performance of the protein was tested in combination with protease (PURAFECT.RTM. or PURAFECT.RTM. Prime) plus amylase (ACE prime described in WO2010/115021 or POWERASE.RTM.). Swatches were cut to 3 cm.times.3 cm in size, read on a Konica Minolta CR-400 reflectometer for pre-wash RGB values, and 4 swatches of each stain type (12 g including ballast soil) were added to each test beaker along with 6 stainless steel balls. Water hardness was adjusted to a final concentration of 100 ppm. The commercially available detergent OMO color powder (Unilever) was heat-inactivated and used at a dose of 5.25 g/L diluted in 50 mM CAPS buffer pH 10.0. The commercially available Persil Small and Mighty bio liquid detergent (Unilever) contained no enzymes and was used without heat-inactivation at a dose of 2.33 g/L diluted in 50 mM HEPES buffer pH 8.2. Varying doses (0.25, 0.5, 1 and 2.5 ppm) of Bsp Man4 fragments along with 0.5 ppm PURAFECT.RTM. Prime and 0.1 ppm ACE prime with liquid detergents and 0.8 ppm PURAFECT.RTM. and 0.2 ppm POWERASE.RTM. with powder detergent were added to each beaker. The washing cycle time was 45 minutes at 40.degree. C. After the wash, the swatches were removed, rinsed for 5 minutes in cold tap water, spun in a laundry centrifuge and laid flat in heating cabinet to dry. The dry swatches were covered with dark cloth at room temperature and stain removal was assessed by measuring the RGB values with a Konica Minolta CR-400 reflectometer. The % SR readings for 0.25 ppm Bsp Man4 fragments are shown in FIG. 6A (OMO color powder detergent) and 6B (Persil Small & Mighty liquid detergent).

Example 7

Comparison of Bsp Man4 to Other Mannanases

A. Identification of Homologous Mannanases

[0253] Homologs were identified by BLAST search (Altschul et al., Nucleic Acids Res. 25:3389-402, 1997) against the NCBI non-redundant protein database (nr) using the amino acid sequence of the mature form of Bsp Man4 (SEQ ID NO:8) as the query sequence. Only sequences with a percent identity of 40% or higher were retained. Percent identity (PID) is defined as the number of identical residues divided by the number of aligned residues in the pairwise alignment. Table 7-1 provides the list of sequences identified having a percent identity of 40% or higher to Bsp Man4. Table 7-1 provides NCBI and SEQ ID NOs. for each homolog, as well as the length (number of amino acids) of each sequence; and the PID (percent identity).

B. Alignment of Homologous Mannanase Sequences

[0254] The sequences of Bsp Man4 and selected homologs were multiply aligned using CLUSTALW software (Thompson et al., Nucleic Acids Res, 22:4673-4680, 1994) using default parameters. The alignment was refined with MUSCLE (MUltiple Sequence Comparison by Log-Expectation, Edgar, Nucleic Acids Res, 32:1792-1797, 2004) using default parameters. For homologous sequences, only regions that correspond to seed sequences are shown. Redundant sequences that are 98% or higher in PID were not included in further analyses. FIG. 7 shows the alignment of Bsp Man4 with homologous mannanases.

C. Phylogenetic Tree

[0255] A phylogenetic tree was built for Bsp Man4 with the Neighbor-Joining algorithm using ClustalW software with 10000 bootstraps based on the refined alignments described above. Bootstrapping was used to assess the reliability of the tree branches (Felsenstein, Evolution 39:783-791, 1985). Other ClustalW parameters were set at the default values. The phylogenetic tree was rendered using the program PhyloWidget: web-based visualizations for the tree of life at www.phylowidget.org (Jordan and Piel, Bioinformatics, 24:1641-1642, 2008). The phyogenetic tree for Bsp Man4 is shown in FIG. 8.

TABLE-US-00009 TABLE 7-1 List of Bsp Man4 Homologs with a Percent Identity of 40 or Greater to the Mature Form of SEQ ID NO: 8 SEQ ID LENGTH % IDENTITY Homolog NO: (# residues) (PID) U.S. Pat. No. 6,566,114-0010 15 586 74.6 ZP-06365324 16 1121 54.2 AAT42241 17 510 54.0 BAE80444 18 997 49.8 ZP_06625371 19 854 47.1 2BVT_A 20 475 44.0 Gte Man1 21 1008 43.3 YP_003850806 22 1410 43.0 ZP_06922280 23 786 42.0 YP_003487354 24 667 41.0

TABLE-US-00010 TABLE 7-2 List of Bsp Man4 Homologs with a Percent Identity of 40 or Greater to the Catalytic Domain (296 residues) of SEQ ID NO: 9 Homolog Length PID(%) U.S. Pat. No. 6,566,114-0010 586 83.4 ZP_06365324 1121 70.8 BAE80444 997 67.2 ZP_06625371 854 66.9 AAT42241 510 64.9 Gte Man1 294 61.6 YP_003850806 1410 55.3 ZP_06922280 786 51 YP_003487354 667 51 2BVT_A 475 50.2

Example 8

Prediction of Functional Domains of Bsp Man4

[0256] The location of functional domains such as the catalytic region and carbohydrate-binding domains of Bsp Man4 was determined using reference sequences within the BLAST result list using the Conserved Domain Search Service (CD Search) tool located in the NCBI web site. CD-Search uses RPS-BLAST (Reverse Position-Specific BLAST) to compare a query sequence against position-specific score matrices that have been prepared from conserved domain alignments present in the Conserved Domain Database (CDD). The results of CD-Search are presented as annotated protein domains on the user query sequence. The protein sequence of homolog D2M1G9 (TrEMBL, former NCBI ZP.sub.--06365324) was entered into the CD Search tool to identify the catalytic and carbohydrate binding domains of Bsp Man4. The amino acid sequence of D2M1G9 shares 54.2% identity with Bsp Man4.

[0257] Functional domains were predicted using ClustalW alignments by AlignX within Vector NTI (Invitrogen). Based on the alignment with D2M1G9, the catalytic domain of Bsp Man4 was predicted to be 296 amino acids in length, starting at position D11 and ending with position W306. The binding module CBM27 was predicted to be 161 amino acids in length, starting at position L493 and ending with position L653. The binding module CBM11 was predicted to be 160 amino acids in length, starting at position L666 and ending with position R825. A complete description of the carbohydrate binding module family classifications can be found in the CAZy carbohydrate active enzymes database (www.cazy.org/Carbohydrate-Binding-Modules.html). Catalytic residues of Bsp Man4 were predicted to be at E179 and E289, using a literature reference describing the structure of Cellulomonas fimi CfMan26A (Le Nours et al., Biochemistry 44:12700-8, 2005). All positions were calculated from the start of the mature protein sequence. FIG. 9 shows the functional domains of BspMan 4.

[0258] The amino acid sequence of the catalytic domain of Bsp Man4 is set forth as SEQ ID NO:9:

TABLE-US-00011 DEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTLTNPAPRTGSTQSE VFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKNTAQSMKVAHDLGG IITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAEFNAWLDNIAALAH ELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRYTVEYLRD VKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDNKDNAGS EAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTLDW.

[0259] Next, a homology model of Bsp Man4 was built by threading the amino acid sequence of Bsp Man4 onto to the three dimensional structure of a Cellulomonas fimi mannanase. The following steps to construct the homology model were accomplished using the program suite "MOE" provided by Chemical Computing Group Inc., (Montreal, Quebec, Canada). The first step involved using the protein sequence of Bsp Man4 to search for homologous sequences of known structures in the Protein Data Bank (www.rcsb.org/pdb/home/home.do). From this search, the Cellulomonas fimi mannanase (pdb entry 2.times.2Y) was identified, and the shared identity between 2.times.2Y and Bsp Man4 was found to be 40.4%. The next step involved threading the sequence of Bsp Man4onto related elements of the known sequence of the Cellulomonas fimi mannanase. The threading process itself includes several constraints. One such constraint involves keeping the main chain and side chain structure of the conserved residues the same. Another constraint involves keeping the main chain atoms fixed, while searching for rotamers of the replaced side chains of non conserved residues which are most compatible with the ensemble of neighboring atoms within the model. When residues were inserted, a loop structure library was used to model the possible insertions. The entire threading process was repeated 10 times with the potential for selecting different rotamers. All models were subjected to limited energy minimization, followed by selection of the model having the lowest energy. Amino acid sequences of truncated species of Bsp Man4, based on the homology model are shown below.

[0260] The amino acid sequence of truncated species 1 of Bsp Man4 is set forth as SEQ ID NO:10.

TABLE-US-00012 RQLNMADEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTLTNPAPRT GSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKNTAQSMKV AHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAEFNAWLDN IAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRYT VEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDN KDNAGSEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTL DWYTRVLDAIAADEDARKISYMLTWANFGWPNNMYVPYRDIHNELGGDHE LLPDFEAFHADDYTAFRDEIKGKIYNTGKEYTVSPHEPFMYVISPITGST VTSETVTIQAKVANDEHARVTFRVDGSSLEEEMVFNDDTLYYTGSFTPDA AVNGGAVDVIVAYYSSGEK.

[0261] The amino acid sequence of truncated species 2 of Bsp Man4 is set forth as SEQ ID NO:11.

TABLE-US-00013 RQLNMADEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTLTNPAPRT GSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKNTAQSMKV AHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAEFNAWLDN IAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRYT VEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDN KDNAGSEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTL DWYTRVLDAIAADEDARKISYMLTWANFGWPNNMYVPYRDIHNELGGDHE LLPDFEAFHADDYTAFRDEIKGKIYNTGKEYTV.

[0262] In addition, the amino acid sequence of the alpha, beta and gamma forms of recombinant Bsp Man4 were determined by Edman degradation and mass spectroscopy.

[0263] The alpha form of Bsp Man4 comprises residues 1-849 of SEQ ID NO:7. The amino acid sequence of the alpha form of Bsp Man4 (MW.about.100 kDa by SDS-PAGE, or MW.about.94 kDa by mass spectroscopy) is set forth as SEQ ID NO:12.

TABLE-US-00014 AGKSQEGRQLNMADEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTL TNPAPRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKN TAQSMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAE FNAWLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQY KAIFRYTVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIF GIDNYDNKDNAGSEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGI NRQGNTLDWYTRVLDAIAADEDARKISYMLTWANFGWPNNMYVPYRDIHN ELGGDHELLPDFEAFHADDYTAFRDEIKGKIYNTGKEYTVSPHEPFMYVI SPITGSTVTSETVTIQAKVANDEHARVTFRVDGSSLEEEMVFNDDTLYYT GSFTPDAAVNGGAVDVIVAYYSSGEKVQEETIRLFVKIPEMSLLTLTFDD DINGIKSNGTWPEDGVTSEIDHAIVDGDGKLMFSVQGMSPTETWQELKLE LTELSDVNIDAVKKMKFDALIPAGSEEGSVQGIVQLPPDWETKYGMNETT KSIKDLETVTVNGSDYKRLEVTVSIDNQGGATGIALSLVGSQLDLLEPVY IDNIELLNSFEAPPADSFLVDDFEGYFGDDTLLHRNYSSNGDPITLSLTS EFKNNGEFGLKYDYSIGSMGYAGRQTSLGPVDWSGANAFEFWMKHGQLEG NHLTVQIRIGDVSFEKNLELMDAHEGVVTIPFSEFAPAAWENKPGVIIDE QKLKRVSQFALYTGGARQSGTIYFDDLRAVYDESLPSVPVPKEEEEEKE.

[0264] The beta form of Bsp Man4 comprises residues 1-669 of SEQ ID NO:7. The amino acid sequence of the beta form of Bsp Man4 (MW.about.70 kDa by SDS-PAGE, or MW.about.74 kDa by mass spectroscopy) is set forth as SEQ ID NO:13.

TABLE-US-00015 AGKSQEGRQLNMADEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTL TNPAPRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKN TAQSMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAE FNAWLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQY KAIFRYTVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIF GIDNYDNKDNAGSEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGI NRQGNTLDWYTRVLDAIAADEDARKISYMLTWANFGWPNNMYVPYRDIHN ELGGDHELLPDFEAFHADDYTAFRDEIKGKIYNTGKEYTVSPHEPFMYVI SPITGSTVTSETVTIQAKVANDEHARVTFRVDGSSLEEEMVFNDDTLYYT GSFTPDAAVNGGAVDVIVAYYSSGEKVQEETIRLFVKIPEMSLLTLTFDD DINGIKSNGTWPEDGVTSEIDHAIVDGDGKLMFSVQGMSPTETWQELKLE LTELSDVNIDAVKKMKFDALIPAGSEEGSVQGIVQLPPDWETKYGMNETT KSIKDLETVTVNGSDYKRLEVTVSIDNQGGATGIALSLVGSQLDLLEPVY IDNIELLNSFEAPPADSFL.

[0265] The gamma form of Bsp Man4 comprises residues 1-494 of SEQ ID NO:7. The amino acid sequence of the gamma form of Bsp Man4 (MW.about.50 kDa by SDS-PAGE, or MW.about.54 kDa by mass spectroscopy) is set forth as SEQ ID NO:14.

TABLE-US-00016 AGKSQEGRQLNMADEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTL TNPAPRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKN TAQSMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAE FNAWLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQY KAIFRYTVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIF GIDNYDNKDNAGSEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGI NRQGNTLDWYTRVLDAIAADEDARKISYMLTWANFGWPNNMYVPYRDIHN ELGGDHELLPDFEAFHADDYTAFRDEIKGKIYNTGKEYTVSPHEPFMYVI SPITGSTVTSETVTIQAKVANDEHARVTFRVDGSSLEEEMVFNDDTLYYT GSFTPDAAVNGGAVDVIVAYYSSGEKVQEETIRLFVKIPEMS.

[0266] In further embodiments, additional truncated forms of Bsp Man4 are provided. One form comprises residues 1 to 350 of SEQ ID NO:8, another form comprises residues 1 to 475 of SEQ ID NO:8, another form comprises residues 1 to 675 of SEQ ID NO:8, and yet another form comprises residues 1 to 850 of SEQ ID NO:8 (as described below).

Example 9

Cloning of the Bacillus sp. Mannanase Bsp Man4 Variants

[0267] Different length of Bacillus sp. mannanase Bsp Man4 variants were obtained by PCR from Bacillus sp. mannanase Bsp Man4 wild type plasmid DNA pZQ186 (aprE-Bsp Man4). Primers were designed based on Bacillus sp. mannanase Bsp Man4 full-length gene sequences and Bsp Man4 Pfam domain structures (The Pfam protein families database: M. Punta, P. C. Coggill, R. Y. Eberhardt, J. Mistry, J. Tate, C. Boursnell, N. Pang, K. Forslund, G. Ceric, J. Clements, A. Heger, L. Holm, E. L. L. Sonnhammer, S. R. Eddy, A. Bateman, R. D. Finn, Nucleic Acids Research (2012) Database Issue 40:D290-D301). The diagrams of the truncations can be found in FIG. 10. Primers used in this study are: For: 5'-ACTAGCCGACTAGTTCACAAGAAGGGCGTCAACTTAAC-3' (SEQ ID NO:25), v1_Rev: 5'-CTTACGGG CTCGAGTTAACCTAATTCATTGTGGATATCACG-3' (SEQ ID NO:26, v2_Rev: 5'-CTTACGGG CTCGAGTTATTGGACTTTTTCTCCACTAGAATAATAAG-3' (SEQ ID NO:27, v3_Rev: 5'-CTTACGGG CTCGAGTTACCCAAAATAACCTTCAAAATCATC-3' (SEQ ID NO:28, v4 Rev: 5'-CTTACGGGCTCGAGTTAAATAGGAGCGACCTCTTTTTCCTCTTC-3' (SEQ ID NO:29). The PCR primers contain Spe I restriction enzyme sites and Xho I restriction enzyme sites for cloning purpose. PCR was performed using a thermocycler with KOD-plus polymerase (TOYOBA) according to the instructions of the manufacturer (annealing temperature of 58.degree. C.). The nucleic acid sequences of PCR products are confirmed by sequencing analysis.

[0268] The PCR products were digested with Spe I and Xho I (New England Biolabs) and then ligated into expression vector p2JM. The ligation mixture was transformed into E. coli TOP10 chemical competent cells following manufacture's protocol (Life Technology). Transformed cells were then plated on Luria Broth agar plates and selected by 50 ppm ampicillin antibiotics, incubated at 37 degree over night. Positive clones containing the correct inserts were confirmed by sequencing analysis.

[0269] The nucleotide sequence of the Bacillus sp. mannanase Bsp Man4v1 gene is set forth as SEQ ID NO: 30.

TABLE-US-00017 (SEQ ID NO: 30) TCACAAGAAGGGCGTCAACTTAACATGGCAGATGAGGATGCTTCAAAGTA TACGAAGGAGTTATTTGCTTTTCTTCAAGATGTAAGTGGTTCACAAGTGT TATTTGGACAACAGCATGCAACAGATGAAGGATTAACTTTAACAAATCCA GCTCCAAGAACAGGTTCCACTCAATCTGAAGTTTTCAATGCAGTTGGGGA TTATCCAGCTGTGTTTGGATGGGACACGAATAGCCTAGATGGTCGTGAAA AGCCTGGCATTGCAGGTAATGTAGAACAAAGTATAAAAAATACGGCTCAG TCCATGAAAGTGGCTCATGATTTAGGAGGGATTATTACACTAAGCATGCA CCCAGATAATTTTGTAACAGGGGGTCCTTATGGTGATACAACAGGGAATG TTGTAAAAGAAATTCTTCCAGGTGGATCAAAACATGCAGAGTTTAACGCG TGGTTGGACAATATTGCTGCGCTTGCTCACGAGCTGAAAGATGAGAATGG TGAACCTATTCCGATGATTTTTCGGCCATTCCATGAACAAACAGGATCTT GGTTTTGGTGGGGAGCAAGCACAACTTCACCCGAACAATATAAAGCGATT TTTCGTTATACAGTAGAATATTTGCGAGATGTTAAAGGCGTAAATAATAT TTTATATGGCTTTTCACCTGGGGCGGGACCTGCTGGAGATGTAAATCGCT ATTTAGAAACATATCCAGGGGATGATTACGTTGATATTTTCGGTATTGAC AATTATGACAATAAAGACAATGCAGGGTCAGAAGCTTGGTTAAGTGGTAT GGTCAAAGACTTGGCGATGATTAGCCGATTAGCTGAACAAAAAGAAAAAG TAGCGGCTTTTACTGAGTATGGGTACAGTGCAACCGGAATTAATCGTCAA GGGAATACATTAGACTGGTACACACGTGTATTAGATGCGATTGCTGCTGA TGAAGACGCACGTAAAATATCATACATGTTGACATGGGCGAACTTTGGTT GGCCGAATAATATGTATGTTCCTTATCGTGATATCCACAATGAATTAGGT TAA

[0270] The amino acid sequence of the Bacillus sp. mannanase Bsp Man4v1 protein is set forth as SEQ ID NO: 31. The signal peptide is shown in italics and lowercase. There is a restriction enzyme site introduced between signal peptide and first codon of Bacillus sp. mannanase Bsp Man4v1, which is shown in lowercase and underline.

TABLE-US-00018 (SEQ ID NO: 31) mrskklwisllfaltliftmafsnmsaqatsSQEGRQLNMADEDASKYTK ELFAFLQDVSGSQVLFGQQHATDEGLTLTNPAPRTGSTQSEVFNAVGDYP AVFGWDTNSLDGREKPGIAGNVEQSIKNTAQSMKVAHDLGGIITLSMHPD NFVTGGPYGDTTGNVVKEILPGGSKHAEFNAWLDNIAALAHELKDENGEP IPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRYTVEYLRDVKGVNNILY GFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDNKDNAGSEAWLSGMVK DLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTLDWYTRVLDAIAADED ARKISYMLTWANFGWPNNMYVPYRDIHNELG

[0271] The amino acid sequence of the mature form of Bsp Man4v1 is set forth as SEQ ID NO: 32.

TABLE-US-00019 (SEQ ID NO: 32) SQEGRQLNMADEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTLTNP APRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKNTAQ SMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAEFNA WLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAI FRYTVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGID NYDNKDNAGSEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQ GNTLDWYTRVLDAIAADEDARKISYMLTWANFGWPNNMYVPYRDIHNELG

[0272] The nucleotide sequence of the Bacillus sp. mannanase Bsp Man4v2 gene is set forth as SEQ ID NO: 33.

TABLE-US-00020 (SEQ ID NO: 33) TCACAAGAAGGGCGTCAACTTAACATGGCAGATGAGGATGCTTCAAAGTA TACGAAGGAGTTATTTGCTTTTCTTCAAGATGTAAGTGGTTCACAAGTGT TATTTGGACAACAGCATGCAACAGATGAAGGATTAACTTTAACAAATCCA GCTCCAAGAACAGGTTCCACTCAATCTGAAGTTTTCAATGCAGTTGGGGA TTATCCAGCTGTGTTTGGATGGGACACGAATAGCCTAGATGGTCGTGAAA AGCCTGGCATTGCAGGTAATGTAGAACAAAGTATAAAAAATACGGCTCAG TCCATGAAAGTGGCTCATGATTTAGGAGGGATTATTACACTAAGCATGCA CCCAGATAATTTTGTAACAGGGGGTCCTTATGGTGATACAACAGGGAATG TTGTAAAAGAAATTCTTCCAGGTGGATCAAAACATGCAGAGTTTAACGCG TGGTTGGACAATATTGCTGCGCTTGCTCACGAGCTGAAAGATGAGAATGG TGAACCTATTCCGATGATTTTTCGGCCATTCCATGAACAAACAGGATCTT GGTTTTGGTGGGGAGCAAGCACAACTTCACCCGAACAATATAAAGCGATT TTTCGTTATACAGTAGAATATTTGCGAGATGTTAAAGGCGTAAATAATAT TTTATATGGCTTTTCACCTGGGGCGGGACCTGCTGGAGATGTAAATCGCT ATTTAGAAACATATCCAGGGGATGATTACGTTGATATTTTCGGTATTGAC AATTATGACAATAAAGACAATGCAGGGTCAGAAGCTTGGTTAAGTGGTAT GGTCAAAGACTTGGCGATGATTAGCCGATTAGCTGAACAAAAAGAAAAAG TAGCGGCTTTTACTGAGTATGGGTACAGTGCAACCGGAATTAATCGTCAA GGGAATACATTAGACTGGTACACACGTGTATTAGATGCGATTGCTGCTGA TGAAGACGCACGTAAAATATCATACATGTTGACATGGGCGAACTTTGGTT GGCCGAATAATATGTATGTTCCTTATCGTGATATCCACAATGAATTAGGT GGAGACCATGAGTTATTACCGGACTTTGAAGCTTTCCATGCGGATGACTA CACAGCATTTCGAGATGAGATAAAAGGAAAGATATATAATACTGGAAAGG AATATACCGTTTCTCCTCATGAGCCGTTTATGTATGTTATATCTCCGATT ACAGGTTCTACAGTGACAAGCGAAACGGTAACAATCCAAGCAAAAGTAGC GAATGACGAACACGCAAGAGTCACTTTCAGGGTCGATGGTTCTAGTTTGG AAGAAGAAATGGTTTTCAATGATGACACTTTATATTATACAGGTTCTTTT ACACCAGATGCAGCAGTGAATGGCGGAGCTGTTGATGTGATTGTAGCTTA TTATTCTAGTGGAGAAAAAGTCCAATAA

[0273] The amino acid sequence of the Bacillus sp. mannanase Bsp Man4v2 protein is set forth as SEQ ID NO: 34. The signal peptide is shown in italics and lowercase. There is a restriction enzyme site introduced between signal peptide and first codon of Bacillus sp. mannanase Bsp Man4v2, which is shown in lowercase and underline.

TABLE-US-00021 (SEQ ID NO: 34) mrskklwisllfaltliftmafsnmsaqatsSQEGRQLNMADEDASKYTK ELFAFLQDVSGSQVLFGQQHATDEGLTLTNPAPRTGSTQSEVFNAVGDYP AVFGWDTNSLDGREKPGIAGNVEQSIKNTAQSMKVAHDLGGIITLSMHPD NFVTGGPYGDTTGNVVKEILPGGSKHAEFNAWLDNIAALAHELKDENGEP IPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRYTVEYLRDVKGVNNILY GFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDNKDNAGSEAWLSGMVK DLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTLDWYTRVLDAIAADED ARKISYMLTWANFGWPNNMYVPYRDIHNELGGDHELLPDFEAFHADDYTA FRDEIKGKIYNTGKEYTVSPHEPFMYVISPITGSTVTSETVTIQAKVAND EHARVTFRVDGSSLEEEMVFNDDTLYYTGSFTPDAAVNGGAVDVIVAYYS SGEKVQ

[0274] The amino acid sequence of the mature form of Bsp Man4v2 is set forth as SEQ ID NO:35.

TABLE-US-00022 (SEQ ID NO: 35) SQEGRQLNMADEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTLTNP APRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKNTAQ SMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAEFNA WLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAI FRYTVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGID NYDNKDNAGSEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQ GNTLDWYTRVLDAIAADEDARKISYMLTWANFGWPNNMYVPYRDIHNELG GDHELLPDFEAFHADDYTAFRDEIKGKIYNTGKEYTVSPHEPFMYVISPI TGSTVTSETVTIQAKVANDEHARVTFRVDGSSLEEEMVFNDDTLYYTGSF TPDAAVNGGAVDVIVAYYSSGEKVQ

[0275] The nucleotide sequence of the Bacillus sp. mannanase Bsp Man4v3 gene is set forth as SEQ ID NO: 36.

TABLE-US-00023 (SEQ ID NO: 36) TCACAAGAAGGGCGTCAACTTAACATGGCAGATGAGGATGCTTCAAAGTA TACGAAGGAGTTATTTGCTTTTCTTCAAGATGTAAGTGGTTCACAAGTGT TATTTGGACAACAGCATGCAACAGATGAAGGATTAACTTTAACAAATCCA GCTCCAAGAACAGGTTCCACTCAATCTGAAGTTTTCAATGCAGTTGGGGA TTATCCAGCTGTGTTTGGATGGGACACGAATAGCCTAGATGGTCGTGAAA AGCCTGGCATTGCAGGTAATGTAGAACAAAGTATAAAAAATACGGCTCAG TCCATGAAAGTGGCTCATGATTTAGGAGGGATTATTACACTAAGCATGCA CCCAGATAATTTTGTAACAGGGGGTCCTTATGGTGATACAACAGGGAATG TTGTAAAAGAAATTCTTCCAGGTGGATCAAAACATGCAGAGTTTAACGCG TGGTTGGACAATATTGCTGCGCTTGCTCACGAGCTGAAAGATGAGAATGG TGAACCTATTCCGATGATTTTTCGGCCATTCCATGAACAAACAGGATCTT GGTTTTGGTGGGGAGCAAGCACAACTTCACCCGAACAATATAAAGCGATT TTTCGTTATACAGTAGAATATTTGCGAGATGTTAAAGGCGTAAATAATAT TTTATATGGCTTTTCACCTGGGGCGGGACCTGCTGGAGATGTAAATCGCT ATTTAGAAACATATCCAGGGGATGATTACGTTGATATTTTCGGTATTGAC AATTATGACAATAAAGACAATGCAGGGTCAGAAGCTTGGTTAAGTGGTAT GGTCAAAGACTTGGCGATGATTAGCCGATTAGCTGAACAAAAAGAAAAAG TAGCGGCTTTTACTGAGTATGGGTACAGTGCAACCGGAATTAATCGTCAA GGGAATACATTAGACTGGTACACACGTGTATTAGATGCGATTGCTGCTGA TGAAGACGCACGTAAAATATCATACATGTTGACATGGGCGAACTTTGGTT GGCCGAATAATATGTATGTTCCTTATCGTGATATCCACAATGAATTAGGT GGAGACCATGAGTTATTACCGGACTTTGAAGCTTTCCATGCGGATGACTA CACAGCATTTCGAGATGAGATAAAAGGAAAGATATATAATACTGGAAAGG AATATACCGTTTCTCCTCATGAGCCGTTTATGTATGTTATATCTCCGATT ACAGGTTCTACAGTGACAAGCGAAACGGTAACAATCCAAGCAAAAGTAGC GAATGACGAACACGCAAGAGTCACTTTCAGGGTCGATGGTTCTAGTTTGG AAGAAGAAATGGTTTTCAATGATGACACTTTATATTATACAGGTTCTTTT ACACCAGATGCAGCAGTGAATGGCGGAGCTGTTGATGTGATTGTAGCTTA TTATTCTAGTGGAGAAAAAGTCCAAGAAGAAACAATTCGTTTATTTGTAA AAATTCCTGAAATGTCTTTGTTAACATTAACGTTTGATGATGATATAAAC GGAATCAAAAGCAATGGAACATGGCCTGAAGATGGTGTAACATCTGAAAT TGACCACGCTATTGTAGATGGAGACGGCAAGTTGATGTTCTCTGTTCAAG GAATGTCACCTACTGAAACATGGCAAGAGCTCAAGTTAGAATTAACAGAA CTATCAGATGTGAACATTGATGCGGTTAAGAAAATGAAGTTTGACGCGCT TATCCCAGCAGGTAGTGAAGAAGGTTCAGTCCAAGGAATCGTACAACTTC CACCGGATTGGGAGACGAAATATGGGATGAATGAAACAACGAAGTCAATA AAAGACTTAGAGACTGTTACTGTTAATGGAAGCGATTATAAACGGTTGGA AGTGACTGTTTCTATCGACAATCAAGGAGGAGCTACAGGAATCGCTTTAT CATTAGTAGGATCCCAACTCGATTTGTTAGAACCTGTCTACATCGATAAT ATTGAACTTCTAAATTCCTTTGAAGCACCACCAGCAGATTCTTTTCTTGT TGATGATTTTGAAGGTTATTTTGGGTAA

[0276] The amino acid sequence of the Bacillus sp. mannanase Bsp Man4v3 protein is set forth as SEQ ID NO: 37. The signal peptide is shown in italics and lowercase. There is a restriction enzyme site introduced between signal peptide and first codon of Bacillus sp. mannanase Bsp Man4v3, which is shown in lowercase and underline.

TABLE-US-00024 (SEQ ID NO: 37) MrskklwisllfaltliftmafsnmsaqatsSQEGRQLNMADEDASKYTKELFAFLQDVSGSQVLFGQ QHATDEGLTLTNPAPRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQ SIKNTAQSMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAEFNA WLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRYTVEY LRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDNKDNAGSEAW LSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTLDWYTRVLDAIAADED ARKISYMLTWANFGWPNNMYVPYRDIHNELGGDHELLPDFEAFHADDYTAFRDEIK GKIYNTGKEYTVSPHEPFMYVISPITGSTVTSETVTIQAKVANDEHARVTFRVDGSSL EEEMVFNDDTLYYTGSFTPDAAVNGGAVDVIVAYYSSGEKVQEETIRLFVKIPEMSL LTLTFDDDINGIKSNGTWPEDGVTSEIDHAIVDGDGKLMFSVQGMSPTETWQELKLE LTELSDVNIDAVKKMKFDALIPAGSEEGSVOGIVOLPPDWETKYGMNETTKSIKDLE TVTVNGSDYKRLEVTVSIDNQGGATGIALSLVGSQLDLLEPVYIDNIELLNSFEAPPA DSFLVDDFEGYFG

[0277] The amino acid sequence of the mature form of Bsp Man4v3 is set forth as SEQ ID NO: 38.

TABLE-US-00025 (SEQ ID NO: 38) SQEGRQLNMADEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTLTNP APRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKNTAQ SMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAEFNA WLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAI FRYTVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGID NYDNKDNAGSEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQ GNTLDWYTRVLDAIAADEDARKISYMLTWANFGWPNNMYVPYRDIHNELG GDHELLPDFEAFHADDYTAFRDEIKGKIYNTGKEYTVSPHEPFMYVISPI TGSTVTSETVTIQAKVANDEHARVTFRVDGSSLEEEMVFNDDTLYYTGSF TPDAAVNGGAVDVIVAYYSSGEKVQEETIRLFVKIPEMSLLTLTFDDDIN GIKSNGTWPEDGVTSEIDHAIVDGDGKLMFSVQGMSPTETWQELKLELTE LSDVNIDAVKKMKFDALIPAGSEEGSVQGIVQLPPDWETKYGMNETTKSI KDLETVTVNGSDYKRLEVTVSIDNQGGATGIALSLVGSQLDLLEPVYIDN IELLNSFEAPPADSFLVDDFEGYFG

[0278] The nucleotide sequence of the Bacillus sp. mannanase Bsp Man4v4 gene is set forth as SEQ ID NO: 39.

TABLE-US-00026 (SEQ ID NO: 39) TCACAAGAAGGGCGTCAACTTAACATGGCAGATGAGGATGCTTCAAAGTA TACGAAGGAGTTATTTGCTTTTCTTCAAGATGTAAGTGGTTCACAAGTGT TATTTGGACAACAGCATGCAACAGATGAAGGATTAACTTTAACAAATCCA GCTCCAAGAACAGGTTCCACTCAATCTGAAGTTTTCAATGCAGTTGGGGA TTATCCAGCTGTGTTTGGATGGGACACGAATAGCCTAGATGGTCGTGAAA AGCCTGGCATTGCAGGTAATGTAGAACAAAGTATAAAAAATACGGCTCAG TCCATGAAAGTGGCTCATGATTTAGGAGGGATTATTACACTAAGCATGCA CCCAGATAATTTTGTAACAGGGGGTCCTTATGGTGATACAACAGGGAATG TTGTAAAAGAAATTCTTCCAGGTGGATCAAAACATGCAGAGTTTAACGCG TGGTTGGACAATATTGCTGCGCTTGCTCACGAGCTGAAAGATGAGAATGG TGAACCTATTCCGATGATTTTTCGGCCATTCCATGAACAAACAGGATCTT GGTTTTGGTGGGGAGCAAGCACAACTTCACCCGAACAATATAAAGCGATT TTTCGTTATACAGTAGAATATTTGCGAGATGTTAAAGGCGTAAATAATAT TTTATATGGCTTTTCACCTGGGGCGGGACCTGCTGGAGATGTAAATCGCT ATTTAGAAACATATCCAGGGGATGATTACGTTGATATTTTCGGTATTGAC AATTATGACAATAAAGACAATGCAGGGTCAGAAGCTTGGTTAAGTGGTAT GGTCAAAGACTTGGCGATGATTAGCCGATTAGCTGAACAAAAAGAAAAAG TAGCGGCTTTTACTGAGTATGGGTACAGTGCAACCGGAATTAATCGTCAA GGGAATACATTAGACTGGTACACACGTGTATTAGATGCGATTGCTGCTGA TGAAGACGCACGTAAAATATCATACATGTTGACATGGGCGAACTTTGGTT GGCCGAATAATATGTATGTTCCTTATCGTGATATCCACAATGAATTAGGT GGAGACCATGAGTTATTACCGGACTTTGAAGCTTTCCATGCGGATGACTA CACAGCATTTCGAGATGAGATAAAAGGAAAGATATATAATACTGGAAAGG AATATACCGTTTCTCCTCATGAGCCGTTTATGTATGTTATATCTCCGATT ACAGGTTCTACAGTGACAAGCGAAACGGTAACAATCCAAGCAAAAGTAGC GAATGACGAACACGCAAGAGTCACTTTCAGGGTCGATGGTTCTAGTTTGG AAGAAGAAATGGTTTTCAATGATGACACTTTATATTATACAGGTTCTTTT ACACCAGATGCAGCAGTGAATGGCGGAGCTGTTGATGTGATTGTAGCTTA TTATTCTAGTGGAGAAAAAGTCCAAGAAGAAACAATTCGTTTATTTGTAA AAATTCCTGAAATGTCTTTGTTAACATTAACGTTTGATGATGATATAAAC GGAATCAAAAGCAATGGAACATGGCCTGAAGATGGTGTAACATCTGAAAT TGACCACGCTATTGTAGATGGAGACGGCAAGTTGATGTTCTCTGTTCAAG GAATGTCACCTACTGAAACATGGCAAGAGCTCAAGTTAGAATTAACAGAA CTATCAGATGTGAACATTGATGCGGTTAAGAAAATGAAGTTTGACGCGCT TATCCCAGCAGGTAGTGAAGAAGGTTCAGTCCAAGGAATCGTACAACTTC CACCGGATTGGGAGACGAAATATGGGATGAATGAAACAACGAAGTCAATA AAAGACTTAGAGACTGTTACTGTTAATGGAAGCGATTATAAACGGTTGGA AGTGACTGTTTCTATCGACAATCAAGGAGGAGCTACAGGAATCGCTTTAT CATTAGTAGGATCCCAACTCGATTTGTTAGAACCTGTCTACATCGATAAT ATTGAACTTCTAAATTCCTTTGAAGCACCACCAGCAGATTCTTTTCTTGT TGATGATTTTGAAGGTTATTTTGGGGATGACACGTTGTTACATCGCAATT ATTCTAGCAATGGAGATCCAATTACACTATCGTTAACAAGTGAGTTTAAA AATAATGGAGAATTTGGATTGAAGTATGATTATTCGATTGGCTCGATGGG TTATGCAGGGAGGCAAACATCACTAGGACCTGTCGATTGGAGCGGAGCTA ATGCTTTTGAATTTTGGATGAAACATGGACAACTTGAAGGGAATCATTTA ACTGTACAAATTCGAATAGGTGATGTTAGCTTTGAAAAAAATCTTGAATT AATGGATGCTCATGAAGGTGTAGTGACAATCCCGTTTTCTGAATTTGCTC CAGCTGCTTGGGAAAATAAGCCTGGCGTTATCATTGACGAACAAAAATTG AAAAGAGTGAGTCAATTTGCTCTTTACACAGGCGGGGCTAGACAATCTGG AACAATCTACTTTGATGATTTACGAGCGGTATATGATGAAAGTTTACCAT CAGTTCCAGTTCCGAAAGAGGAGGAAGAGGAAAAAGAGGTCGCTCCTATT TAA

[0279] The amino acid sequence of the Bacillus sp. mannanase Bsp Man4v4 protein is set forth as SEQ ID NO.40. The signal peptide is shown in italics and lowercase. There is a restriction enzyme site introduced between signal peptide and first codon of Bacillus sp. mannanase Bsp Man4v4, which is shown in lowercase and underline.

TABLE-US-00027 (SEQ ID NO: 40) MrskklwisllfaltliftmafsnmsaqatsSQEGRQLNMADEDASKYTKELFAFLQDVSGSQVLFGQ QHATDEGLTLTNPAPRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQ SIKNTAQSMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAEFNA WLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRYTVEY LRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDNKDNAGSEAW LSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTLDWYTRVLDAIAADED ARKISYMLTWANFGWPNNMYVPYRDIHNELGGDHELLPDFEAFHADDYTAFRDEIK GKIYNTGKEYTVSPHEPFMYVISPITGSTVTSETVTIQAKVANDEHARVTFRVDGSSL EEEMVFNDDTLYYTGSFTPDAAVNGGAVDVIVAYYSSGEKVQEETIRLFVKIPEMSL LTLTFDDDINGIKSNGTWPEDGVTSEIDHAIVDGDGKLMFSVQGMSPTETWQELKLE LTELSDVNIDAVKKMKFDALIPAGSEEGSVQGIVQLPPDWETKYGMNETTKSIKDLE TVTVNGSDYKRLEVTVSIDNQGGATGIALSLVGSQLDLLEPVYIDNIELLNSFEAPPA DSFLVDDFEGYFGDDTLLHRNYSSNGDPITLSLTSEFKNNGEFGLKYDYSIGSMGYA GRQTSLGPVDWSGANAFEFWMKHGQLEGNHLTVQIRIGDVSFEKNLELMDAHEGV VTIPFSEFAPAAWENKPGVIIDEQKLKRVSQFALYTGGARQSGTIYFDDLRAVYDESL PSVPVPKEEEEEKEVAPI

[0280] The amino acid sequence of the mature form of Bsp Man4v4 is set forth as SEQ ID NO: 41.

TABLE-US-00028 (SEQ ID NO: 41) SQEGRQLNMADEDASKYTKELFAFLQDVSGSQVLFGQQHATDEGLTLTNP APRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAGNVEQSIKNTAQ SMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHAEFNA WLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAI FRYTVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGID NYDNKDNAGSEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQ GNTLDWYTRVLDAIAADEDARKISYMLTWANFGWPNNMYVPYRDIHNELG GDHELLPDFEAFHADDYTAFRDEIKGKIYNTGKEYTVSPHEPFMYVISPI TGSTVTSETVTIQAKVANDEHARVTFRVDGSSLEEEMVFNDDTLYYTGSF TPDAAVNGGAVDVIVAYYSSGEKVQEETIRLFVKIPEMSLLTLTFDDDIN GIKSNGTWPEDGVTSEIDHAIVDGDGKLMFSVQGMSPTETWQELKLELTE LSDVNIDAVKKMKFDALIPAGSEEGSVQGIVQLPPDWETKYGMNETTKSI KDLETVTVNGSDYKRLEVTVSIDNQGGATGIALSLVGSQLDLLEPVYIDN IELLNSFEAPPADSFLVDDFEGYFGDDTLLHRNYSSNGDPITLSLTSEFK NNGEFGLKYDYSIGSMGYAGRQTSLGPVDWSGANAFEFWMKHGQLEGNHL TVQIRIGDVSFEKNLELMDAHEGVVTIPFSEFAPAAWENKPGVIIDEQKL KRVSQFALYTGGARQSGTIYFDDLRAVYDESLPSVPVPKEEEEEKEVAPI

Example 10

Expression of Bsp Man4 Deletion Variants

[0281] Bsp Man4v1, Bsp Man4v2, Bsp Man4v3 and Bsp Man4v4 PCR products were cloned into p2JM expression vector and the resulting plasmid were labeled as pLL007 (aprE-Bsp Man4 1-350), pLL008 (aprE-Bsp Man4 1-475), pLL009 (aprE-Bsp Man4 1-675) and pLL010 (aprE-Bsp Man4 1-850). Plasmid maps are provided in FIG. 11. The sequence of the deletion version of genes was confirmed by DNA sequencing.

[0282] The plasmid pLL007 (aprE-Bsp Man4 1-350), pLL008 (aprE-Bsp Man4 1-475), pLL009 (aprE-Bsp Man4 1-675) and pLL010 (aprE-Bsp Man4 1-850) are amplified using rolling circle kit (GE Healthcare Life Sciences, NJ) before transformations. Bacillus subtilis (degUHy32, AnprB, Avpr, Aepr, AscoC, AwprA, Ampr, AispA, Abpr) were transformed with the amplified plasmid. The transformed cells were then plated on Luria Agar plates supplemented with 10 ppm kanamycin. Single colony were picked and cultured in shake flasks.

[0283] The nucleotide sequence of Bsp Man4 v1 gene from expression plasmid pLL007 (aprE-Bsp Man4 1-350) is set forth as SEQ ID NO:42. The signal sequence is shown in bold.

TABLE-US-00029 (SEQ ID NO: 42) GTGAGAAGCAAAAAATTGTGGATCAGCTTGTTGTTTGCGTTAACGTTAAT CTTTACGATGGCGTTCAGCAACATGAGCGCGCAGGCAGCTGGTAAAACTA GTTCACAAGAAGGGCGTCAACTTAACATGGCAGATGAGGATGCTTCAAAG TATACGAAGGAGTTATTTGCTTTTCTTCAAGATGTAAGTGGTTCACAAGT GTTATTTGGACAACAGCATGCAACAGATGAAGGATTAACTTTAACAAATC CAGCTCCAAGAACAGGTTCCACTCAATCTGAAGTTTTCAATGCAGTTGGG GATTATCCAGCTGTGTTTGGATGGGACACGAATAGCCTAGATGGTCGTGA AAAGCCTGGCATTGCAGGTAATGTAGAACAAAGTATAAAAAATACGGCTC AGTCCATGAAAGTGGCTCATGATTTAGGAGGGATTATTACACTAAGCATG CACCCAGATAATTTTGTAACAGGGGGTCCTTATGGTGATACAACAGGGAA TGTTGTAAAAGAAATTCTTCCAGGTGGATCAAAACATGCAGAGTTTAACG CGTGGTTGGACAATATTGCTGCGCTTGCTCACGAGCTGAAAGATGAGAAT GGTGAACCTATTCCGATGATTTTTCGGCCATTCCATGAACAAACAGGATC TTGGTTTTGGTGGGGAGCAAGCACAACTTCACCCGAACAATATAAAGCGA TTTTTCGTTATACAGTAGAATATTTGCGAGATGTTAAAGGCGTAAATAAT ATTTTATATGGCTTTTCACCTGGGGCGGGACCTGCTGGAGATGTAAATCG CTATTTAGAAACATATCCAGGGGATGATTACGTTGATATTTTCGGTATTG ACAATTATGACAATAAAGACAATGCAGGGTCAGAAGCTTGGTTAAGTGGT ATGGTCAAAGACTTGGCGATGATTAGCCGATTAGCTGAACAAAAAGAAAA AGTAGCGGCTTTTACTGAGTATGGGTACAGTGCAACCGGAATTAATCGTC AAGGGAATACATTAGACTGGTACACACGTGTATTAGATGCGATTGCTGCT GATGAAGACGCACGTAAAATATCATACATGTTGACATGGGCGAACTTTGG TTGGCCGAATAATATGTATGTTCCTTATCGTGATATCCACAATGAATTAG GTTAA

[0284] The amino acid of Bsp Man4 v1 protein from expression plasmid pLL007 (aprE-Bsp Man4 1-350) is set forth as SEQ ID NO:43. The signal sequence is shown in bold.

TABLE-US-00030 (SEQ ID NO: 43) vrskklwisllfaltliftmafsnmsaqaAGKTSSQEGRQLNMADEDASKYTKELFAFLQDVSGSQV LFGQQHATDEGLTLTNPAPRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAG NVEQSIKNTAQSMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHA EFNAWLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRY TVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDNKDNAG SEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTLDWYTRVLDAIA ADEDARKISYMLTWANFGWPNNMYVPYRDIHNELG

[0285] The nucleotide sequence of Bsp Man4 v2 gene from expression plasmid pLL008 (aprE-Bsp Man4 1-475) is set forth as SEQ ID NO:44. The signal sequence is shown in bold.

TABLE-US-00031 (SEQ ID NO: 44) GTGAGAAGCAAAAAATTGTGGATCAGCTTGTTGTTTGCGTTAACGTTAAT CTTTACGATGGCGTTCAGCAACATGAGCGCGCAGGCAGCTGGTAAAACTA GTTCACAAGAAGGGCGTCAACTTAACATGGCAGATGAGGATGCTTCAAAG TATACGAAGGAGTTATTTGCTTTTCTTCAAGATGTAAGTGGTTCACAAGT GTTATTTGGACAACAGCATGCAACAGATGAAGGATTAACTTTAACAAATC CAGCTCCAAGAACAGGTTCCACTCAATCTGAAGTTTTCAATGCAGTTGGG GATTATCCAGCTGTGTTTGGATGGGACACGAATAGCCTAGATGGTCGTGA AAAGCCTGGCATTGCAGGTAATGTAGAACAAAGTATAAAAAATACGGCTC AGTCCATGAAAGTGGCTCATGATTTAGGAGGGATTATTACACTAAGCATG CACCCAGATAATTTTGTAACAGGGGGTCCTTATGGTGATACAACAGGGAA TGTTGTAAAAGAAATTCTTCCAGGTGGATCAAAACATGCAGAGTTTAACG CGTGGTTGGACAATATTGCTGCGCTTGCTCACGAGCTGAAAGATGAGAAT GGTGAACCTATTCCGATGATTTTTCGGCCATTCCATGAACAAACAGGATC TTGGTTTTGGTGGGGAGCAAGCACAACTTCACCCGAACAATATAAAGCGA TTTTTCGTTATACAGTAGAATATTTGCGAGATGTTAAAGGCGTAAATAAT ATTTTATATGGCTTTTCACCTGGGGCGGGACCTGCTGGAGATGTAAATCG CTATTTAGAAACATATCCAGGGGATGATTACGTTGATATTTTCGGTATTG ACAATTATGACAATAAAGACAATGCAGGGTCAGAAGCTTGGTTAAGTGGT ATGGTCAAAGACTTGGCGATGATTAGCCGATTAGCTGAACAAAAAGAAAA AGTAGCGGCTTTTACTGAGTATGGGTACAGTGCAACCGGAATTAATCGTC AAGGGAATACATTAGACTGGTACACACGTGTATTAGATGCGATTGCTGCT GATGAAGACGCACGTAAAATATCATACATGTTGACATGGGCGAACTTTGG TTGGCCGAATAATATGTATGTTCCTTATCGTGATATCCACAATGAATTAG GTGGAGACCATGAGTTATTACCGGACTTTGAAGCTTTCCATGCGGATGAC TACACAGCATTTCGAGATGAGATAAAAGGAAAGATATATAATACTGGAAA GGAATATACCGTTTCTCCTCATGAGCCGTTTATGTATGTTATATCTCCGA TTACAGGTTCTACAGTGACAAGCGAAACGGTAACAATCCAAGCAAAAGTA GCGAATGACGAACACGCAAGAGTCACTTTCAGGGTCGATGGTTCTAGTTT GGAAGAAGAAATGGTTTTCAATGATGACACTTTATATTATACAGGTTCTT TTACACCAGATGCAGCAGTGAATGGCGGAGCTGTTGATGTGATTGTAGCT TATTATTCTAGTGGAGAAAAAGTCCAATAA

[0286] The amino acid of Bsp Man4 v2 protein from expression plasmid pLL008 (aprE-Bsp Man4 1-475) is set forth as SEQ ID NO:45. The signal sequence is shown in bold.

TABLE-US-00032 (SEQ ID NO: 45) vrskklwisllfaltliftmafsnmsaqaAGKTSSQEGRQLNMADEDASKYTKELFAFLQDVSGSQV LFGQQHATDEGLTLTNPAPRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAG NVEQSIKNTAQSMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHA EFNAWLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRY TVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDNKDNAG SEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTLDWYTRVLDAIA ADEDARKISYMLTWANFGWPNNMYVPYRDIHNELGGDHELLPDFEAFHADDYTAF RDEIKGKIYNTGKEYTVSPHEPFMYVISPITGSTVTSETVTIQAKVANDEHARVTFRV DGSSLEEEMVFNDDTLYYTGSFTPDAAVNGGAVDVIVAYYSSGEKVQ

[0287] The nucleotide sequence of Bsp Man4 v3 gene from expression plasmid pLL009 (aprE-Bsp Man4 1-675) is set forth as SEQ ID NO:46. The signal sequence is shown in bold.

TABLE-US-00033 (SEQ ID NO: 46) GTGAGAAGCAAAAAATTGTGGATCAGCTTGTTGTTTGCGTTAACGTTAAT CTTTACGATGGCGTTCAGCAACATGAGCGCGCAGGCAGCTGGTAAAACTA GTTCACAAGAAGGGCGTCAACTTAACATGGCAGATGAGGATGCTTCAAAG TATACGAAGGAGTTATTTGCTTTTCTTCAAGATGTAAGTGGTTCACAAGT GTTATTTGGACAACAGCATGCAACAGATGAAGGATTAACTTTAACAAATC CAGCTCCAAGAACAGGTTCCACTCAATCTGAAGTTTTCAATGCAGTTGGG GATTATCCAGCTGTGTTTGGATGGGACACGAATAGCCTAGATGGTCGTGA AAAGCCTGGCATTGCAGGTAATGTAGAACAAAGTATAAAAAATACGGCTC AGTCCATGAAAGTGGCTCATGATTTAGGAGGGATTATTACACTAAGCATG CACCCAGATAATTTTGTAACAGGGGGTCCTTATGGTGATACAACAGGGAA TGTTGTAAAAGAAATTCTTCCAGGTGGATCAAAACATGCAGAGTTTAACG CGTGGTTGGACAATATTGCTGCGCTTGCTCACGAGCTGAAAGATGAGAAT GGTGAACCTATTCCGATGATTTTTCGGCCATTCCATGAACAAACAGGATC TTGGTTTTGGTGGGGAGCAAGCACAACTTCACCCGAACAATATAAAGCGA TTTTTCGTTATACAGTAGAATATTTGCGAGATGTTAAAGGCGTAAATAAT ATTTTATATGGCTTTTCACCTGGGGCGGGACCTGCTGGAGATGTAAATCG CTATTTAGAAACATATCCAGGGGATGATTACGTTGATATTTTCGGTATTG ACAATTATGACAATAAAGACAATGCAGGGTCAGAAGCTTGGTTAAGTGGT ATGGTCAAAGACTTGGCGATGATTAGCCGATTAGCTGAACAAAAAGAAAA AGTAGCGGCTTTTACTGAGTATGGGTACAGTGCAACCGGAATTAATCGTC AAGGGAATACATTAGACTGGTACACACGTGTATTAGATGCGATTGCTGCT GATGAAGACGCACGTAAAATATCATACATGTTGACATGGGCGAACTTTGG TTGGCCGAATAATATGTATGTTCCTTATCGTGATATCCACAATGAATTAG GTGGAGACCATGAGTTATTACCGGACTTTGAAGCTTTCCATGCGGATGAC TACACAGCATTTCGAGATGAGATAAAAGGAAAGATATATAATACTGGAAA GGAATATACCGTTTCTCCTCATGAGCCGTTTATGTATGTTATATCTCCGA TTACAGGTTCTACAGTGACAAGCGAAACGGTAACAATCCAAGCAAAAGTA GCGAATGACGAACACGCAAGAGTCACTTTCAGGGTCGATGGTTCTAGTTT GGAAGAAGAAATGGTTTTCAATGATGACACTTTATATTATACAGGTTCTT TTACACCAGATGCAGCAGTGAATGGCGGAGCTGTTGATGTGATTGTAGCT TATTATTCTAGTGGAGAAAAAGTCCAAGAAGAAACAATTCGTTTATTTGT AAAAATTCCTGAAATGTCTTTGTTAACATTAACGTTTGATGATGATATAA ACGGAATCAAAAGCAATGGAACATGGCCTGAAGATGGTGTAACATCTGAA ATTGACCACGCTATTGTAGATGGAGACGGCAAGTTGATGTTCTCTGTTCA AGGAATGTCACCTACTGAAACATGGCAAGAGCTCAAGTTAGAATTAACAG AACTATCAGATGTGAACATTGATGCGGTTAAGAAAATGAAGTTTGACGCG CTTATCCCAGCAGGTAGTGAAGAAGGTTCAGTCCAAGGAATCGTACAACT TCCACCGGATTGGGAGACGAAATATGGGATGAATGAAACAACGAAGTCAA TAAAAGACTTAGAGACTGTTACTGTTAATGGAAGCGATTATAAACGGTTG GAAGTGACTGTTTCTATCGACAATCAAGGAGGAGCTACAGGAATCGCTTT ATCATTAGTAGGATCCCAACTCGATTTGTTAGAACCTGTCTACATCGATA ATATTGAACTTCTAAATTCCTTTGAAGCACCACCAGCAGATTCTTTTCTT GTTGATGATTTTGAAGGTTATTTTGGGTAA

[0288] The amino acid of Bsp Man4 v3 protein from expression plasmid pLL009 (aprE-Bsp Man4 1-675) is set forth as SEQ ID NO:47. The signal sequence is shown in bold.

TABLE-US-00034 (SEQ ID NO: 47) vrskklwisllfaltliftmafsnmsaqaAGKTSSQEGRQLNMADEDASKYTKELFAFLQDVSGSQV LFGQQHATDEGLTLTNPAPRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAG NVEQSIKNTAQSMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHA EFNAWLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRY TVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDNKDNAG SEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTLDWYTRVLDAIA ADEDARKISYMLTWANFGWPNNMYVPYRDIHNELGGDHELLPDFEAFHADDYTAF RDEIKGKIYNTGKEYTVSPHEPFMYVISPITGSTVTSETVTIQAKVANDEHARVTFRV DGSSLEEEMVFNDDTLYYTGSFTPDAAVNGGAVDVIVAYYSSGEKVQEETIRLFVKI PEMSLLTLTFDDDINGIKSNGTWPEDGVTSEIDHAIVDGDGKLMFSVQGMSPTETWQ ELKLELTELSDVNIDAVKKMKFDALIPAGSEEGSVQGIVQLPPDWETKYGMNETTKS IKDLETVTVNGSDYKRLEVTVSIDNQGGATGIALSLVGSQLDLLEPVYIDNIELLNSFE APPADSFLVDDFEGYFG

[0289] The nucleotide sequence of Bsp Man4 v4 gene from expression plasmid pLL010 (aprE-Bsp Man4 1-850) is set forth as SEQ ID NO:48. The signal sequence is shown in bold.

TABLE-US-00035 (SEQ ID NO: 48) GTGAGAAGCAAAAAATTGTGGATCAGCTTGTTGTTTGCGTTAACGTTAAT CTTTACGATGGCGTTCAGCAACATGAGCGCGCAGGCAGCTGGTAAAACTA GTTCACAAGAAGGGCGTCAACTTAACATGGCAGATGAGGATGCTTCAAAG TATACGAAGGAGTTATTTGCTTTTCTTCAAGATGTAAGTGGTTCACAAGT GTTATTTGGACAACAGCATGCAACAGATGAAGGATTAACTTTAACAAATC CAGCTCCAAGAACAGGTTCCACTCAATCTGAAGTTTTCAATGCAGTTGGG GATTATCCAGCTGTGTTTGGATGGGACACGAATAGCCTAGATGGTCGTGA AAAGCCTGGCATTGCAGGTAATGTAGAACAAAGTATAAAAAATACGGCTC AGTCCATGAAAGTGGCTCATGATTTAGGAGGGATTATTACACTAAGCATG CACCCAGATAATTTTGTAACAGGGGGTCCTTATGGTGATACAACAGGGAA TGTTGTAAAAGAAATTCTTCCAGGTGGATCAAAACATGCAGAGTTTAACG CGTGGTTGGACAATATTGCTGCGCTTGCTCACGAGCTGAAAGATGAGAAT GGTGAACCTATTCCGATGATTTTTCGGCCATTCCATGAACAAACAGGATC TTGGTTTTGGTGGGGAGCAAGCACAACTTCACCCGAACAATATAAAGCGA TTTTTCGTTATACAGTAGAATATTTGCGAGATGTTAAAGGCGTAAATAAT ATTTTATATGGCTTTTCACCTGGGGCGGGACCTGCTGGAGATGTAAATCG CTATTTAGAAACATATCCAGGGGATGATTACGTTGATATTTTCGGTATTG ACAATTATGACAATAAAGACAATGCAGGGTCAGAAGCTTGGTTAAGTGGT ATGGTCAAAGACTTGGCGATGATTAGCCGATTAGCTGAACAAAAAGAAAA AGTAGCGGCTTTTACTGAGTATGGGTACAGTGCAACCGGAATTAATCGTC AAGGGAATACATTAGACTGGTACACACGTGTATTAGATGCGATTGCTGCT GATGAAGACGCACGTAAAATATCATACATGTTGACATGGGCGAACTTTGG TTGGCCGAATAATATGTATGTTCCTTATCGTGATATCCACAATGAATTAG GTGGAGACCATGAGTTATTACCGGACTTTGAAGCTTTCCATGCGGATGAC TACACAGCATTTCGAGATGAGATAAAAGGAAAGATATATAATACTGGAAA GGAATATACCGTTTCTCCTCATGAGCCGTTTATGTATGTTATATCTCCGA TTACAGGTTCTACAGTGACAAGCGAAACGGTAACAATCCAAGCAAAAGTA GCGAATGACGAACACGCAAGAGTCACTTTCAGGGTCGATGGTTCTAGTTT GGAAGAAGAAATGGTTTTCAATGATGACACTTTATATTATACAGGTTCTT TTACACCAGATGCAGCAGTGAATGGCGGAGCTGTTGATGTGATTGTAGCT TATTATTCTAGTGGAGAAAAAGTCCAAGAAGAAACAATTCGTTTATTTGT AAAAATTCCTGAAATGTCTTTGTTAACATTAACGTTTGATGATGATATAA ACGGAATCAAAAGCAATGGAACATGGCCTGAAGATGGTGTAACATCTGAA ATTGACCACGCTATTGTAGATGGAGACGGCAAGTTGATGTTCTCTGTTCA AGGAATGTCACCTACTGAAACATGGCAAGAGCTCAAGTTAGAATTAACAG AACTATCAGATGTGAACATTGATGCGGTTAAGAAAATGAAGTTTGACGCG CTTATCCCAGCAGGTAGTGAAGAAGGTTCAGTCCAAGGAATCGTACAACT TCCACCGGATTGGGAGACGAAATATGGGATGAATGAAACAACGAAGTCAA TAAAAGACTTAGAGACTGTTACTGTTAATGGAAGCGATTATAAACGGTTG GAAGTGACTGTTTCTATCGACAATCAAGGAGGAGCTACAGGAATCGCTTT ATCATTAGTAGGATCCCAACTCGATTTGTTAGAACCTGTCTACATCGATA ATATTGAACTTCTAAATTCCTTTGAAGCACCACCAGCAGATTCTTTTCTT GTTGATGATTTTGAAGGTTATTTTGGGGATGACACGTTGTTACATCGCAA TTATTCTAGCAATGGAGATCCAATTACACTATCGTTAACAAGTGAGTTTA AAAATAATGGAGAATTTGGATTGAAGTATGATTATTCGATTGGCTCGATG GGTTATGCAGGGAGGCAAACATCACTAGGACCTGTCGATTGGAGCGGAGC TAATGCTTTTGAATTTTGGATGAAACATGGACAACTTGAAGGGAATCATT TAACTGTACAAATTCGAATAGGTGATGTTAGCTTTGAAAAAAATCTTGAA TTAATGGATGCTCATGAAGGTGTAGTGACAATCCCGTTTTCTGAATTTGC TCCAGCTGCTTGGGAAAATAAGCCTGGCGTTATCATTGACGAACAAAAAT TGAAAAGAGTGAGTCAATTTGCTCTTTACACAGGCGGGGCTAGACAATCT GGAACAATCTACTTTGATGATTTACGAGCGGTATATGATGAAAGTTTACC ATCAGTTCCAGTTCCGAAAGAGGAGGAAGAGGAAAAAGAGGTCGCTCCTA TTTAA

[0290] The amino acid of Bsp Man4 v4 protein from expression plasmid pLL009 (aprE-Bsp Man4 1-850) is set forth as SEQ ID NO:49. The signal sequence is shown in bold.

TABLE-US-00036 (SEQ ID NO: 49) vrskklwisllfaltliftmafsnmsaqaAGKTSSQEGRQLNMADEDASKYTKELFAFLQDVSGSQV LFGQQHATDEGLTLTNPAPRTGSTQSEVFNAVGDYPAVFGWDTNSLDGREKPGIAG NVEQSIKNTAQSMKVAHDLGGIITLSMHPDNFVTGGPYGDTTGNVVKEILPGGSKHA EFNAWLDNIAALAHELKDENGEPIPMIFRPFHEQTGSWFWWGASTTSPEQYKAIFRY TVEYLRDVKGVNNILYGFSPGAGPAGDVNRYLETYPGDDYVDIFGIDNYDNKDNAG SEAWLSGMVKDLAMISRLAEQKEKVAAFTEYGYSATGINRQGNTLDWYTRVLDAIA ADEDARKISYMLTWANFGWPNNMYVPYRDIHNELGGDHELLPDFEAFHADDYTAF RDEIKGKIYNTGKEYTVSPHEPFMYVISPITGSTVTSETVTIQAKVANDEHARVTFRV DGSSLEEEMVFNDDTLYYTGSFTPDAAVNGGAVDVIVAYYSSGEKVQEETIRLFVKI PEMSLLTLTFDDDINGIKSNGTWPEDGVTSEIDHAIVDGDGKLMFSVQGMSPTETWQ ELKLELTELSDVNIDAVKKMKFDALIPAGSEEGSVQGIVQLPPDWETKYGMNETTKS IKDLETVTVNGSDYKRLEVTVSIDNQGGATGIALSLVGSQLDLLEPVYIDNIELLNSFE APPADSFLVDDFEGYFGDDTLLHRNYSSNGDPITLSLTSEFKNNGEFGLKYDYSIGSM GYAGRQTSLGPVDWSGANAFEFWMKHGQLEGNHLTVQIRIGDVSFEKNLELMDAH EGVVTIPFSEFAPAAWENKPGVIIDEQKLKRVSQFALYTGGARQSGTIYFDDLRAVYD ESLPSVPVPKEEEEEKEVAPI

Example 11

Purification of Bsp Man4v2

[0291] Bsp Man4v2 was purified via the hydrophobic interaction and anion-exchange chromatography. Ammonium sulphate was added to 700 mL crude broth from the shake flask to a final concentration of 1 M. The solution was then loaded onto a 150 mL phenyl-sepharose FF (high sub) XK 26/20 column pre-equilibrated with 20 mM sodium phosphate pH 6.0 (buffer A) with 1 M ammonium sulphate (buffer B). The target protein was eluted with 100%-0 buffer B in 3 CV, followed by 3 CV of MilliQ H.sub.2O. The fractions containing target protein were pooled and desalted by ultrafiltration. The desalted sample was then loaded onto a 150 ml Q-Sepharose FF XK26/20 column pre-equilibrated with 20 mM Tris-HCl, pH 7.5 (buffer C). After sample loading, the column was washed with the same buffer for 2 column volumes, followed by a gradient of 0-80% buffer C with 1 M NaCl (buffer D) in 8 CVs. The fractions containing target protein were pooled and concentrated using 10K Amicon Ultra-15 devices. The sample was above 95% pure and stored in 40% glycerol at -80.degree. C. until usage.

Example 12

Mannanase Activity of Bsp Man4 and Bsp Man4v2

[0292] The beta 1-4 mannanase activity of Bsp Man4 and Bsp Man4v2 was measured using 1% Galactomannan (Carob; Low Viscosity) (P-GALML; Lot 10501) purchased from Megazyme International Ireland (Bray, Ireland). The assay was performed in 50 mM sodium acetate pH 5.0, 0.005% Tween-80 buffer at 50.degree. C. for 10 minutes or in 50 mM HEPES pH 8.2, 0.005% Tween-80 buffer at 30.degree. C. for 30 minutes. The released reducing sugar was quantified in a PAHBAH (p-Hydroxy benzoic acid hydrazide) assay (Lever, Anal. Biochem. 47:248, 1972). A standard curve using mannose was generated and used to calculate enzyme activity units. In this assay, one mannanase unit is defined as the amount of enzyme required to generate 1 micromole of mannose reducing sugar equivalents per minute under the conditions of the assay. The specific activity of purified Bsp Man4 at pH 5.0 was determined to be 67 units/mg, whereas Bsp Man4v2 was determined to be 156 units/mg towards low viscosity carob galactomannan using the above method.

Example 13

pH Profiles of Bsp Man4v2

[0293] The pH profiles of Bsp Man4v2 were determined using the beta-mannazyme tablet assay from Megazyme (Tmnz 1/02; Azurine-crosslinked carob galactomannan) with minor modifications to the suggested protocol. The assay was performed in 50 mm Acetate/Bis-Tris/HEPES/CHES buffer adjusted to pH values between 4 and 11. The enzyme solution was diluted into the assay buffer and 500 .mu.l of the enzyme solution was equilibrated at 55.degree. C. before adding one substrate tablet. After 10 minutes, the reaction was stopped by adding 10 ml 2% Tris pH 12. The tubes were left at room temperature for 5 minutes, stirred and the liquid filtered through a Whatman No.1 paper filter. Release of blue dye from the substrate was quantified by measuring the optical density at 590 nm. Enzyme activity at each pH was reported as relative activity where the activity at the pH optimum was set to 100%. The pH profile of Bsp Man4v2 is shown in FIG. 12.

Example 14

[0294] Temperature Profiles of Bsp Man4 and Bsp Man4v2 The temperature optimum of purified Bsp Man4v2 was determined by assaying for mannanase activity using the beta-mannazyme tablet assay from Megazyme (Tmnz 1/02; Azurine-crosslinked carob galactomannan) with minor modifications to the suggested protocol. The assay was performed at temperatures varying between 40.degree. C. and 69.degree. C. for 10 minutes in 50 mM HEPES buffer at pH 8.2. The activity was reported as relative activity where the activity at the temperature optimum was set to 100%. The temperature profile of Bsp Man4v2 is shown in FIG. 13.

Example 15

Thermostability of Bsp Man4 and Bsp Man4v2

[0295] The thermostability of Bsp Man4 and Bsp Man4v2 was determined in 50 mM HEPES buffer pH 8.2. The enzymes were incubated at desired temperature for 2 hours in the Bio-Rad PCR machine. The remaining activity of the samples was measured using the Azo-Carob Galactomannan assay from Megazyme (ACGLM 03/07; Remazolbrilliant Blue R dyed carob galactomannan) with minor modifications to the suggested protocol. The activity of the sample kept on ice was defined as 100% activity. The thermostability results for Bsp Man4 and Bsp Man4v2 are shown in FIG. 14. At temperatures lower than 55.degree. C., no activity loss was detected for either Bsp Man4 or Bsp Man4v2 during a 2-hour incubation. Bsp Man4v2 retains more activity than Bsp Man4 at the elevated temperatures.

Example 16

Liquid Laundry Detergent Compositions Comprising Bsp Man4

[0296] In this example, various formulations for liquid laundry detergent compositions are provided. In each of these formulations, Bsp Man4 is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.

TABLE-US-00037 TABLE 16-1 Liquid Laundry Detergent Compositions Formulations Compound I II III IV V LAS 24.0 32.0 6.0 3.0 6.0 NaC.sub.16-C.sub.17HSAS -- -- -- 5.0 -- C.sub.12-C.sub.15AE.sub.1.8S -- -- 8.0 7.0 5.0 C.sub.8-C.sub.10 propyl 2.0 2.0 2.0 2.0 1.0 dimethyl amine C.sub.12-C.sub.14 alkyl dimethyl -- -- -- -- 2.0 amine oxide C.sub.12-C.sub.15AS -- -- 17.0 -- 8.0 CFAA -- 5.0 4.0 4.0 3.0 C.sub.12-C.sub.14 Fatty alcohol 12.0 6.0 1.0 1.0 1.0 ethoxylate C.sub.12-C.sub.18 Fatty acid 3.0 -- 4.0 2.0 3.0 Citric acid (anhydrous) 4.5 5.0 3.0 2.0 1.0 DETPMP -- -- 1.0 1.0 0.5 Monoethanolamine 5.0 5.0 5.0 5.0 2.0 Sodium hydroxide -- -- 2.5 1.0 1.5 1N HCl aqueous solution #1 #1 -- -- -- Propanediol 12.7 14.5 13.1 10. 8.0 Ethanol 1.8 2.4 4.7 5.4 1.0 DTPA 0.5 0.4 0.3 0.4 0.5 Pectin Lyase -- -- -- 0.005 -- Amylase 0.001 0.002 -- -- Cellulase -- -- 0.0002 0.0001 Lipase 0.1 -- 0.1 -- 0.1 NprE (optional) 0.05 0.3 -- 0.5 0.2 PMN -- -- 0.08 -- -- Protease A (optional) -- -- -- -- 0.1 Aldose Oxidase -- -- 0.3 -- 0.003 ZnCl2 0.1 0.05 0.05 0.05 0.02 Ca formate 0.05 0.07 0.05 0.06 0.07 DETBCHD -- -- 0.02 0.01 -- SRP1 0.5 0.5 -- 0.3 0.3 Boric acid -- -- -- -- 2.4 Sodium xylene sulfonate -- -- 3.0 -- -- Sodium cumene -- -- -- 0.3 0.5 sulfonate DC 3225C 1.0 1.0 1.0 1.0 1.0 2-butyl-octanol 0.03 0.04 0.04 0.03 0.03 Brightener 1 0.12 0.10 0.18 0.08 0.10 Balance to 100% perfume/dye and/or water #1: Add 1N HCl aq. soln to adjust the neat pH of the formula in the range from about 3 to about 5. The pH of Examples 16(I)-(II) is about 5 to about 7, and of 16(III)-(V) is about 7.5 to about 8.5.

Example 17

Liquid Hand Dishwashing Detergent Compositions Comprising Bsp Man4

[0297] In this example, various hand dish liquid detergent formulations are provided. In each of these formulations, Bsp Man4 is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.

TABLE-US-00038 TABLE 17-1 Liquid Hand Dishwashing Detergent Compositions Formulations Compound I II III IV V VI C.sub.12-C.sub.15AE.sub.1.8S 30.0 28.0 25.0 -- 15.0 10.0 LAS -- -- -- 5.0 15.0 12.0 Paraffin Sulfonate -- -- -- 20.0 -- -- C.sub.10-C.sub.18 Alkyl 5.0 3.0 7.0 -- -- -- Dimethyl Amine Oxide Betaine 3.0 -- 1.0 3.0 1.0 -- C.sub.12 poly-OH fatty -- -- -- 3.0 -- 1.0 acid amide C.sub.14 poly-OH fatty -- 1.5 -- -- -- -- acid amide C.sub.11E.sub.9 2.0 -- 4.0 -- -- 20.0 DTPA -- -- -- -- 0.2 -- Tri-sodium Citrate 0.25 -- -- 0.7 -- -- dehydrate Diamine 1.0 5.0 7.0 1.0 5.0 7.0 MgCl.sub.2 0.25 -- -- 1.0 -- -- nprE (optional) 0.02 0.01 -- 0.01 -- 0.05 PMN -- -- 0.03 -- 0.02 -- Protease A (optional) -- 0.01 -- -- -- -- Amylase 0.001 -- -- 0.002 -- 0.001 Aldose Oxidase 0.03 -- 0.02 -- 0.05 -- Sodium Cumene -- -- -- 2.0 1.5 3.0 Sulphonate PAAC 0.01 0.01 0.02 -- -- -- DETBCHD -- -- -- 0.01 0.02 0.01 Balance to 100% perfume/dye and/or water The pH of Examples 17(I)-(VI) is about 8 to about 11.

Example 18

Liquid Automatic Dishwashing Detergent Compositions Comprising Bsp Man4

[0298] In this example, various liquid automatic dishwashing detergent formulations are provided. In each of these formulations, Bsp Man4 polypeptide is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.

TABLE-US-00039 TABLE 18-1 Liquid Automatic Dishwashing Detergent Compositions Formulations Compound I II III IV V STPP 16 16 18 16 16 Potassium Sulfate -- 10 8 -- 10 1,2 propanediol 6.0 0.5 2.0 6.0 0.5 Boric Acid -- -- -- 4.0 3.0 CaCl.sub.2 dihydrate 0.04 0.04 0.04 0.04 0.04 Nonionic 0.5 0.5 0.5 0.5 0.5 nprE (optional) 0.1 0.03 -- 0.03 -- PMN -- -- 0.05 -- 0.06 Protease B (optional) -- -- -- 0.01 -- Amylase 0.02 -- 0.02 0.02 -- Aldose Oxidase -- 0.15 0.02 -- 0.01 Galactose Oxidase -- -- 0.01 -- 0.01 PAAC 0.01 -- -- 0.01 -- DETBCHD -- 0.01 -- -- 0.01 Balance to 100% perfume/dye and/or water

Example 19

Granular and/or Tablet Laundry Compositions Comprising Bsp Man4

[0299] This example provides various formulations for granular and/or tablet laundry detergents. In each of these formulations, Bsp Man4 is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.

TABLE-US-00040 TABLE 19-1 Granular and/or Tablet Laundry Compositions Formulations Compound I II III IV V Base Product C.sub.14-C.sub.15AS or TAS 8.0 5.0 3.0 3.0 3.0 LAS 8.0 -- 8.0 -- 7.0 C.sub.12-C.sub.15AE.sub.3S 0.5 2.0 1.0 -- -- C.sub.12-C.sub.15E.sub.5 or E.sub.3 2.0 -- 5.0 2.0 2.0 QAS -- -- -- 1.0 1.0 Zeolite A 20.0 18.0 11.0 -- 10.0 SKS-6 (dry add) -- -- 9.0 -- -- MA/AA 2.0 2.0 2.0 -- -- AA -- -- -- -- 4.0 3Na Citrate 2H.sub.2O -- 2.0 -- -- -- Citric Acid 2.0 -- 1.5 2.0 -- (Anhydrous) DTPA 0.2 0.2 -- -- -- EDDS -- -- 0.5 0.1 -- HEDP -- -- 0.2 0.1 -- PB1 3.0 4.8 -- -- 4.0 Percarbonate -- -- 3.8 5.2 -- NOBS 1.9 -- -- -- -- NACA OBS -- -- 2.0 -- -- TAED 0.5 2.0 2.0 5.0 1.00 BB1 0.06 -- 0.34 -- 0.14 BB2 -- 0.14 -- 0.20 -- Anhydrous Na 15.0 18.0 -- 15.0 15.0 Carbonate Sulfate 5.0 12.0 5.0 17.0 3.0 Silicate -- 1.0 -- -- 8.0 nprE (optional) 0.03 -- 0.1 0.06 -- PMN -- 0.05 -- -- 0.1 Protease B (optional) -- 0.01 -- -- -- Protease C (optional) -- -- -- 0.01 -- Lipase -- 0.008 -- -- -- Amylase 0.001 -- -- -- 0.001 Cellulase -- 0.0014 -- -- -- Pectin Lyase 0.001 0.001 0.001 0.001 0.001 Aldose Oxidase 0.03 -- 0.05 -- -- PAAC -- 0.01 -- -- 0.05 Balance to 100% Moisture and/or Minors* *Perfume, dye, brightener/SRP1/Na carboxymethylcellulose/photobleach/MgSO.sub.4/PVPVI/suds suppressor/high molecular PEG/clay.

Example 20

Additional Liquid Laundry Detergents Comprising Bsp Man4

[0300] This example provides further formulations for liquid laundry detergents. In each of these formulations, Bsp Man4 is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.

TABLE-US-00041 TABLE 20-1 Liquid Laundry Detergents Formulations Compound IA IB II III IV V LAS 11.5 11.5 9.0 -- 4.0 -- C.sub.12-C.sub.15AE.sub.2.85S -- -- 3.0 18.0 -- 16.0 C.sub.14-C.sub.15E.sub.2.5S 11.5 11.5 3.0 -- 16.0 -- C.sub.12-C.sub.13E.sub.9 -- -- 3.0 2.0 2.0 1.0 C.sub.12-C.sub.13E.sub.7 3.2 3.2 -- -- -- -- CFAA -- -- -- 5.0 -- 3.0 TPKFA 2.0 2.0 -- 2.0 0.5 2.0 Citric Acid (Anhy.) 3.2 3.2 0.5 1.2 2.0 1.2 Ca formate 0.1 0.1 0.06 0.1 -- -- Na formate 0.5 0.5 0.06 0.1 0.05 0.05 ZnCl2 0.1 0.05 0.06 0.03 0.05 0.05 Na Culmene Sulfonate 4.0 4.0 1.0 3.0 1.2 -- Borate 0.6 0.6 1.5 -- -- -- Na Hydroxide 6.0 6.0 2.0 3.5 4.0 3.0 Ethanol 2.0 2.0 1.0 4.0 4.0 3.0 1,2 Propanediol 3.0 3.0 2.0 8.0 8.0 5.0 Monoethanoaminel 3.0 3.0 1.5 1.0 2.5 1.0 TEPAE 2.0 2.0 -- 1.0 1.0 1.0 nprE (optional) 0.03 0.05 -- 0.03 -- 0.02 PMN -- -- 0.01 -- 0.08 -- Protease A (optional) -- -- 0.01 -- -- -- Lipase -- -- -- 0.002 -- -- Amylase -- -- -- -- 0.002 -- Cellulase -- -- -- -- -- 0.0001 Pectin Lyase 0.005 0.005 -- -- -- Aldose Oxidase 0.05 -- -- 0.05 -- 0.02 Galactose oxidase -- 0.04 PAAC 0.03 0.03 0.02 -- -- -- DETBCHD -- -- -- 0.02 0.01 -- SRP 1 0.2 0.2 -- 0.1 -- -- DTPA -- -- -- 0.3 -- -- PVNO -- -- -- 0.3 -- 0.2 Brightener 1 0.2 0.2 0.07 0.1 -- -- Silicone antifoam 0.04 0.04 0.02 0.1 0.1 0.1 Balance to 100% perfume/dye and/or water

Example 21

High Density Dishwashing Detergents Comprising Bsp Man4

[0301] This example provides various formulations for high density dishwashing detergents. In each of these compact formulations, Bsp Man4 is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.

TABLE-US-00042 TABLE 21-1 High Density Dishwashing Detergents Formulations Compound I II III IV V VI STPP -- 45.0 45.0 -- -- 40.0 3Na Citrate 17.0 -- -- 50.0 40.2 -- 2H.sub.2O Na Carbonate 17.5 14.0 20.0 -- 8.0 33.6 Bicarbonate -- -- -- 26.0 -- -- Silicate 15.0 15.0 8.0 -- 25.0 3.6 Metasilicate 2.5 4.5 4.5 -- -- -- PB1 -- -- 4.5 -- -- -- PB4 -- -- -- 5.0 -- -- Percarbonate -- -- -- -- -- 4.8 BB1 -- 0.1 0.1 -- 0.5 -- BB2 0.2 0.05 -- 0.1 -- 0.6 Nonionic 2.0 1.5 1.5 3.0 1.9 5.9 HEDP 1.0 -- -- -- -- -- DETPMP 0.6 -- -- -- -- -- PAAC 0.03 0.05 0.02 -- -- -- Paraffin 0.5 0.4 0.4 0.6 -- -- nprE (optional) 0.072 0.053 -- 0.026 -- 0.01 PMN -- -- 0.053 -- 0.059 -- Protease B -- -- -- -- -- 0.01 (optional) Amylase 0.012 -- 0.012 -- 0.021 0.006 Lipase -- 0.001 -- 0.005 -- -- Pectin Lyase 0.001 0.001 0.001 -- -- -- Aldose Oxidase 0.05 0.05 0.03 0.01 0.02 0.01 BTA 0.3 0.2 0.2 0.3 0.3 0.3 Polycarboxy- 6.0 -- -- -- 4.0 0.9 late Perfume 0.2 0.1 0.1 0.2 0.2 0.2 Balance to 100% Moisture and/or Minors* *Brightener/dye/SRP1/Na carboxymethylcellulose/photobleach/MgSO.sub.4/PVPVI/suds suppressor/high molecular PEG/clay. The pH of Examples 21(I) through (VI) is from about 9.6 to about 11.3.

Example 22

Tablet Dishwashing Detergent Compositions Comprising Bsp Man4

[0302] This example provides various tablet dishwashing detergent formulations. The following tablet detergent compositions of the present disclosure are prepared by compression of a granular dishwashing detergent composition at a pressure of 13KN/cm.sup.2 using a standard 12 head rotary press. In each of these formulations, Bsp Man4 is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.

TABLE-US-00043 TABLE 22-1 Tablet Dishwashing Detergent Compositions Formulations Compound I II III IV V VI VII VIII STPP -- 48.8 44.7 38.2 -- 42.4 46.1 46.0 3Na Citrate 2H.sub.2O 20.0 -- -- -- 35.9 -- -- -- Na Carbonate 20.0 5.0 14.0 15.4 8.0 23.0 20.0 -- Silicate 15.0 14.8 15.0 12.6 23.4 2.9 4.3 4.2 Lipase 0.001 -- 0.01 -- 0.02 -- -- -- Protease B 0.01 -- -- -- -- -- -- -- (optional) Protease C -- -- -- -- -- 0.01 -- -- (optional) nprE (optional) 0.01 0.08 -- 0.04 -- 0.023 -- 0.05 PMN -- -- 0.05 -- 0.052 -- 0.023 -- Amylase 0.012 0.012 0.012 -- 0.015 -- 0.017 0.002 Pectin Lyase 0.005 -- -- 0.002 -- -- -- -- Aldose Oxidase -- 0.03 -- 0.02 0.02 -- 0.03 -- PB1 -- -- 3.8 -- 7.8 -- -- 4.5 Percarbonate 6.0 -- -- 6.0 -- 5.0 -- -- BB1 0.2 -- 0.5 -- 0.3 0.2 -- -- BB2 -- 0.2 -- 0.5 -- -- 0.1 0.2 Nonionic 1.5 2.0 2.0 2.2 1.0 4.2 4.0 6.5 PAAC 0.01 0.01 0.02 -- -- -- -- -- DETBCHD -- -- -- 0.02 0.02 -- -- -- TAED -- -- -- -- -- 2.1 -- 1.6 HEDP 1.0 -- -- 0.9 -- 0.4 0.2 -- DETPMP 0.7 -- -- -- -- -- -- -- Paraffin 0.4 0.5 0.5 0.5 -- -- 0.5 -- BTA 0.2 0.3 0.3 0.3 0.3 0.3 0.3 -- Polycarboxylate 4.0 -- -- -- 4.9 0.6 0.8 -- PEG 400-30,000 -- -- -- -- -- 2.0 -- 2.0 Glycerol -- -- -- -- -- 0.4 -- 0.5 Perfume -- -- -- 0.05 0.2 0.2 0.2 0.2 Balance to 100% Moisture and/or Minors* *Brightener/SRP1/Na carboxymethylcellulose/photobleach/MgSO.sub.4/PVPVI/suds suppressor/high molecular PEG/clay. The pH of Examples 22(I) through 22(VII) is from about 10 to about 11.5; pH of 22(VIII) is from 8-10. The tablet weight of Examples 22(I) through 22(VIII) is from about 20 grams to about 30 grams.

Example 23

Liquid Hard Surface Cleaning Detergents Comprising Bsp Man4

[0303] This example provides various formulations for liquid hard surface cleaning detergents. In each of these formulations, Bsp Man4 is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.

TABLE-US-00044 TABLE 23-1 Liquid Hard Surface Cleaning Detergents Formulations Compound I II III IV V VI VII C.sub.9-C.sub.11E.sub.5 2.4 1.9 2.5 2.5 2.5 2.4 2.5 C.sub.12-C.sub.14E.sub.5 3.6 2.9 2.5 2.5 2.5 3.6 2.5 C.sub.7-C.sub.9E.sub.6 -- -- -- -- 8.0 -- -- C.sub.12-C.sub.14E.sub.21 1.0 0.8 4.0 2.0 2.0 1.0 2.0 LAS -- -- -- 0.8 0.8 -- 0.8 Sodium culmene 1.5 2.6 -- 1.5 1.5 1.5 1.5 sulfonate Isachem .RTM. AS 0.6 0.6 -- -- -- 0.6 -- Na.sub.2CO.sub.3 0.6 0.13 0.6 0.1 0.2 0.6 0.2 3Na Citrate 2H.sub.2O 0.5 0.56 0.5 0.6 0.75 0.5 0.75 NaOH 0.3 0.33 0.3 0.3 0.5 0.3 0.5 Fatty Acid 0.6 0.13 0.6 0.1 0.4 0.6 0.4 2-butyl octanol 0.3 0.3 -- 0.3 0.3 0.3 0.3 PEG DME-2000 .RTM. 0.4 -- 0.3 0.35 0.5 -- -- PVP 0.3 0.4 0.6 0.3 0.5 -- -- MME PEG (2000) .RTM. -- -- -- -- -- 0.5 0.5 Jeffamine .RTM. ED-2001 -- 0.4 -- -- 0.5 -- -- PAAC -- -- -- 0.03 0.03 0.03 -- DETBCHD 0.03 0.05 0.05 -- -- -- -- nprE (optional) 0.07 -- 0.08 0.03 -- 0.01 0.04 PMN -- 0.05 -- -- 0.06 -- -- Protease B (optional) -- -- -- -- -- 0.01 -- Amylase 0.12 0.01 0.01 -- 0.02 -- 0.01 Lipase -- 0.001 -- 0.005 -- 0.005 -- Pectin Lyase 0.001 -- 0.001 -- -- -- 0.002 ZnCl2 0.02 0.01 0.03 0.05 0.1 0.05 0.02 Calcium Formate 0.03 0.03 0.01 -- -- -- -- PB1 -- 4.6 -- 3.8 -- -- -- Aldose Oxidase 0.05 -- 0.03 -- 0.02 0.02 0.05 Balance to 100% perfume/dye and/or water The pH of Examples 23(I) through (VII) is from about 7.4 to about 9.5.

Sequence CWU 1

1

4913273DNABacillus sp. SWT81 1atgggaacat ggaaaaaggg gtttgtgtta tttattgtcc taatgttagt ttttgatgta 60tcgatgttgg gtgtaaatgt aagcgcttca caagaagggc gtcaacttaa catggcagat 120gaggatgctt caaagtatac gaaggagtta tttgcttttc ttcaagatgt aagtggttca 180caagtgttat ttggacaaca gcatgcaaca gatgaaggat taactttaac aaatccagct 240ccaagaacag gttccactca atctgaagtt ttcaatgcag ttggggatta tccagctgtg 300tttggatggg acacgaatag cctagatggt cgtgaaaagc ctggcattgc aggtaatgta 360gaacaaagta taaaaaatac ggctcagtcc atgaaagtgg ctcatgattt aggagggatt 420attacactaa gcatgcaccc agataatttt gtaacagggg gtccttatgg tgatacaaca 480gggaatgttg taaaagaaat tcttccaggt ggatcaaaac atgcagagtt taacgcgtgg 540ttggacaata ttgctgcgct tgctcacgag ctgaaagatg agaatggtga acctattccg 600atgatttttc ggccattcca tgaacaaaca ggatcttggt tttggtgggg agcaagcaca 660acttcacccg aacaatataa agcgattttt cgttatacag tagaatattt gcgagatgtt 720aaaggcgtaa ataatatttt atatggcttt tcacctgggg cgggacctgc tggagatgta 780aatcgctatt tagaaacata tccaggggat gattacgttg atattttcgg tattgacaat 840tatgacaata aagacaatgc agggtcagaa gcttggttaa gtggtatggt caaagacttg 900gcgatgatta gccgattagc tgaacaaaaa gaaaaagtag cggcttttac tgagtatggg 960tacagtgcaa ccggaattaa tcgtcaaggg aatacattag actggtacac acgtgtatta 1020gatgcgattg ctgctgatga agacgcacgt aaaatatcat acatgttgac atgggcgaac 1080tttggttggc cgaataatat gtatgttcct tatcgtgata tccacaatga attaggtgga 1140gaccatgagt tattaccgga ctttgaagct ttccatgcgg atgactacac agcatttcga 1200gatgagataa aaggaaagat atataatact ggaaaggaat ataccgtttc tcctcatgag 1260ccgtttatgt atgttatatc tccgattaca ggttctacag tgacaagcga aacggtaaca 1320atccaagcaa aagtagcgaa tgacgaacac gcaagagtca ctttcagggt cgatggttct 1380agtttggaag aagaaatggt tttcaatgat gacactttat attatacagg ttcttttaca 1440ccagatgcag cagtgaatgg cggagctgtt gatgtgattg tagcttatta ttctagtgga 1500gaaaaagtcc aagaagaaac aattcgttta tttgtaaaaa ttcctgaaat gtctttgtta 1560acattaacgt ttgatgatga tataaacgga atcaaaagca atggaacatg gcctgaagat 1620ggtgtaacat ctgaaattga ccacgctatt gtagatggag acggcaagtt gatgttctct 1680gttcaaggaa tgtcacctac tgaaacatgg caagagctca agttagaatt aacagaacta 1740tcagatgtga acattgatgc ggttaagaaa atgaagtttg acgcgcttat cccagcaggt 1800agtgaagaag gttcagtcca aggaatcgta caacttccac cggattggga gacgaaatat 1860gggatgaatg aaacaacgaa gtcaataaaa gacttagaga ctgttactgt taatggaagc 1920gattataaac ggttggaagt gactgtttct atcgacaatc aaggaggagc tacaggaatc 1980gctttatcat tagtaggatc ccaactcgat ttgttagaac ctgtctacat cgataatatt 2040gaacttctaa attcctttga agcaccacca gcagattctt ttcttgttga tgattttgaa 2100ggttattttg gggatgacac gttgttacat cgcaattatt ctagcaatgg agatccaatt 2160acactatcgt taacaagtga gtttaaaaat aatggagaat ttggattgaa gtatgattat 2220tcgattggct cgatgggtta tgcagggagg caaacatcac taggacctgt cgattggagc 2280ggagctaatg cttttgaatt ttggatgaaa catggacaac ttgaagggaa tcatttaact 2340gtacaaattc gaataggtga tgttagcttt gaaaaaaatc ttgaattaat ggatgctcat 2400gaaggtgtag tgacaatccc gttttctgaa tttgctccag ctgcttggga aaataagcct 2460ggcgttatca ttgacgaaca aaaattgaaa agagtgagtc aatttgctct ttacacaggc 2520ggggctagac aatctggaac aatctacttt gatgatttac gagcggtata tgatgaaagt 2580ttaccatcag ttccagttcc gaaagaggag gaagaggaaa aagaggtcgc tcctattatt 2640tatcattttg aatctggaat tgataattgg gaagggggac aagcaacaca tagcaatggg 2700cacctcaaag taacggttcg tttaggtgaa ggtcagcaaa ccgaagtgaa gaaaacatca 2760aattataatt taacagggta taattatata gtagctaata taaaacatga cgatacagga 2820atgtttggta gtgacccgct tcaagtgaaa atctttacga aagcaggagg ttgggtatgg 2880gctgattcag gaaatcaacc gatttactcc gacgattata ctcaagttgt gtatgatatt 2940actactttag ctaacaaaaa tgcagtccaa gaaatcgggt ttgaattttt ggctccttca 3000ggttcttcag ggacgacgaa tcctttcata gattcagtag cgattgttac gagtctcgat 3060caattgtctg agcagccaga gcagccagaa caaccaggaa caccagatac tgatgataat 3120aaagaggata aagatagaag aaatgtagaa gtgaacgagg aaggacaaaa actacccaaa 3180acagcaacgt caatatttaa ttatttgcta attggttttg tttttgtagg gattggattt 3240agtctattta tttataaaag aagaaaaaca gtg 327321091PRTBacillus sp. SWT81 2Met Gly Thr Trp Lys Lys Gly Phe Val Leu Phe Ile Val Leu Met Leu 1 5 10 15 Val Phe Asp Val Ser Met Leu Gly Val Asn Val Ser Ala Ser Gln Glu 20 25 30 Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys Tyr Thr Lys 35 40 45 Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln Val Leu Phe 50 55 60 Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr Asn Pro Ala 65 70 75 80 Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala Val Gly Asp 85 90 95 Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp Gly Arg Glu 100 105 110 Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys Asn Thr Ala 115 120 125 Gln Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile Thr Leu Ser 130 135 140 Met His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly Asp Thr Thr 145 150 155 160 Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys His Ala Glu 165 170 175 Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His Glu Leu Lys 180 185 190 Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro Phe His Glu 195 200 205 Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr Ser Pro Glu 210 215 220 Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu Arg Asp Val 225 230 235 240 Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly Ala Gly Pro 245 250 255 Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly Asp Asp Tyr 260 265 270 Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp Asn Ala Gly 275 280 285 Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala Met Ile Ser 290 295 300 Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr Glu Tyr Gly 305 310 315 320 Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu Asp Trp Tyr 325 330 335 Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala Arg Lys Ile 340 345 350 Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn Asn Met Tyr 355 360 365 Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly Gly Asp His Glu Leu 370 375 380 Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp Tyr Thr Ala Phe Arg 385 390 395 400 Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly Lys Glu Tyr Thr Val 405 410 415 Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser Pro Ile Thr Gly Ser 420 425 430 Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala Lys Val Ala Asn Asp 435 440 445 Glu His Ala Arg Val Thr Phe Arg Val Asp Gly Ser Ser Leu Glu Glu 450 455 460 Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr Thr Gly Ser Phe Thr 465 470 475 480 Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp Val Ile Val Ala Tyr 485 490 495 Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu Thr Ile Arg Leu Phe Val 500 505 510 Lys Ile Pro Glu Met Ser Leu Leu Thr Leu Thr Phe Asp Asp Asp Ile 515 520 525 Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro Glu Asp Gly Val Thr Ser 530 535 540 Glu Ile Asp His Ala Ile Val Asp Gly Asp Gly Lys Leu Met Phe Ser 545 550 555 560 Val Gln Gly Met Ser Pro Thr Glu Thr Trp Gln Glu Leu Lys Leu Glu 565 570 575 Leu Thr Glu Leu Ser Asp Val Asn Ile Asp Ala Val Lys Lys Met Lys 580 585 590 Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu Glu Gly Ser Val Gln Gly 595 600 605 Ile Val Gln Leu Pro Pro Asp Trp Glu Thr Lys Tyr Gly Met Asn Glu 610 615 620 Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr Val Thr Val Asn Gly Ser 625 630 635 640 Asp Tyr Lys Arg Leu Glu Val Thr Val Ser Ile Asp Asn Gln Gly Gly 645 650 655 Ala Thr Gly Ile Ala Leu Ser Leu Val Gly Ser Gln Leu Asp Leu Leu 660 665 670 Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu Leu Asn Ser Phe Glu Ala 675 680 685 Pro Pro Ala Asp Ser Phe Leu Val Asp Asp Phe Glu Gly Tyr Phe Gly 690 695 700 Asp Asp Thr Leu Leu His Arg Asn Tyr Ser Ser Asn Gly Asp Pro Ile 705 710 715 720 Thr Leu Ser Leu Thr Ser Glu Phe Lys Asn Asn Gly Glu Phe Gly Leu 725 730 735 Lys Tyr Asp Tyr Ser Ile Gly Ser Met Gly Tyr Ala Gly Arg Gln Thr 740 745 750 Ser Leu Gly Pro Val Asp Trp Ser Gly Ala Asn Ala Phe Glu Phe Trp 755 760 765 Met Lys His Gly Gln Leu Glu Gly Asn His Leu Thr Val Gln Ile Arg 770 775 780 Ile Gly Asp Val Ser Phe Glu Lys Asn Leu Glu Leu Met Asp Ala His 785 790 795 800 Glu Gly Val Val Thr Ile Pro Phe Ser Glu Phe Ala Pro Ala Ala Trp 805 810 815 Glu Asn Lys Pro Gly Val Ile Ile Asp Glu Gln Lys Leu Lys Arg Val 820 825 830 Ser Gln Phe Ala Leu Tyr Thr Gly Gly Ala Arg Gln Ser Gly Thr Ile 835 840 845 Tyr Phe Asp Asp Leu Arg Ala Val Tyr Asp Glu Ser Leu Pro Ser Val 850 855 860 Pro Val Pro Lys Glu Glu Glu Glu Glu Lys Glu Val Ala Pro Ile Ile 865 870 875 880 Tyr His Phe Glu Ser Gly Ile Asp Asn Trp Glu Gly Gly Gln Ala Thr 885 890 895 His Ser Asn Gly His Leu Lys Val Thr Val Arg Leu Gly Glu Gly Gln 900 905 910 Gln Thr Glu Val Lys Lys Thr Ser Asn Tyr Asn Leu Thr Gly Tyr Asn 915 920 925 Tyr Ile Val Ala Asn Ile Lys His Asp Asp Thr Gly Met Phe Gly Ser 930 935 940 Asp Pro Leu Gln Val Lys Ile Phe Thr Lys Ala Gly Gly Trp Val Trp 945 950 955 960 Ala Asp Ser Gly Asn Gln Pro Ile Tyr Ser Asp Asp Tyr Thr Gln Val 965 970 975 Val Tyr Asp Ile Thr Thr Leu Ala Asn Lys Asn Ala Val Gln Glu Ile 980 985 990 Gly Phe Glu Phe Leu Ala Pro Ser Gly Ser Ser Gly Thr Thr Asn Pro 995 1000 1005 Phe Ile Asp Ser Val Ala Ile Val Thr Ser Leu Asp Gln Leu Ser 1010 1015 1020 Glu Gln Pro Glu Gln Pro Glu Gln Pro Gly Thr Pro Asp Thr Asp 1025 1030 1035 Asp Asn Lys Glu Asp Lys Asp Arg Arg Asn Val Glu Val Asn Glu 1040 1045 1050 Glu Gly Gln Lys Leu Pro Lys Thr Ala Thr Ser Ile Phe Asn Tyr 1055 1060 1065 Leu Leu Ile Gly Phe Val Phe Val Gly Ile Gly Phe Ser Leu Phe 1070 1075 1080 Ile Tyr Lys Arg Arg Lys Thr Val 1085 1090 343DNAArtificial Sequencesynthetic primer 3tgagcgcgca ggcagctggt aaatcacaag aagggcgtca act 43432DNAArtificial Sequencesynthetic primer 4cgcctcgagt tacactgttt ttcttctttt at 3253282DNAArtificial Sequencesynthetic BSP Man4 gene 5gtgagaagca aaaaattgtg gatcagcttg ttgtttgcgt taacgttaat ctttacgatg 60gcgttcagca acatgagcgc gcaggcagct ggtaaatcac aagaagggcg tcaacttaac 120atggcagatg aggatgcttc aaagtatacg aaggagttat ttgcttttct tcaagatgta 180agtggttcac aagtgttatt tggacaacag catgcaacag atgaaggatt aactttaaca 240aatccagctc caagaacagg ttccactcaa tctgaagttt tcaatgcagt tggggattat 300ccagctgtgt ttggatggga cacgaatagc ctagatggtc gtgaaaagcc tggcattgca 360ggtaatgtag aacaaagtat aaaaaatacg gctcagtcca tgaaagtggc tcatgattta 420ggagggatta ttacactaag catgcaccca gataattttg taacaggggg tccttatggt 480gatacaacag ggaatgttgt aaaagaaatt cttccaggtg gatcaaaaca tgcagagttt 540aacgcgtggt tggacaatat tgctgcgctt gctcacgagc tgaaagatga gaatggtgaa 600cctattccga tgatttttcg gccattccat gaacaaacag gatcttggtt ttggtgggga 660gcaagcacaa cttcacccga acaatataaa gcgatttttc gttatacagt agaatatttg 720cgagatgtta aaggcgtaaa taatatttta tatggctttt cacctggggc gggacctgct 780ggagatgtaa atcgctattt agaaacatat ccaggggatg attacgttga tattttcggt 840attgacaatt atgacaataa agacaatgca gggtcagaag cttggttaag tggtatggtc 900aaagacttgg cgatgattag ccgattagct gaacaaaaag aaaaagtagc ggcttttact 960gagtatgggt acagtgcaac cggaattaat cgtcaaggga atacattaga ctggtacaca 1020cgtgtattag atgcgattgc tgctgatgaa gacgcacgta aaatatcata catgttgaca 1080tgggcgaact ttggttggcc gaataatatg tatgttcctt atcgtgatat ccacaatgaa 1140ttaggtggag accatgagtt attaccggac tttgaagctt tccatgcgga tgactacaca 1200gcatttcgag atgagataaa aggaaagata tataatactg gaaaggaata taccgtttct 1260cctcatgagc cgtttatgta tgttatatct ccgattacag gttctacagt gacaagcgaa 1320acggtaacaa tccaagcaaa agtagcgaat gacgaacacg caagagtcac tttcagggtc 1380gatggttcta gtttggaaga agaaatggtt ttcaatgatg acactttata ttatacaggt 1440tcttttacac cagatgcagc agtgaatggc ggagctgttg atgtgattgt agcttattat 1500tctagtggag aaaaagtcca agaagaaaca attcgtttat ttgtaaaaat tcctgaaatg 1560tctttgttaa cattaacgtt tgatgatgat ataaacggaa tcaaaagcaa tggaacatgg 1620cctgaagatg gtgtaacatc tgaaattgac cacgctattg tagatggaga cggcaagttg 1680atgttctctg ttcaaggaat gtcacctact gaaacatggc aagagctcaa gttagaatta 1740acagaactat cagatgtgaa cattgatgcg gttaagaaaa tgaagtttga cgcgcttatc 1800ccagcaggta gtgaagaagg ttcagtccaa ggaatcgtac aacttccacc ggattgggag 1860acgaaatatg ggatgaatga aacaacgaag tcaataaaag acttagagac tgttactgtt 1920aatggaagcg attataaacg gttggaagtg actgtttcta tcgacaatca aggaggagct 1980acaggaatcg ctttatcatt agtaggatcc caactcgatt tgttagaacc tgtctacatc 2040gataatattg aacttctaaa ttcctttgaa gcaccaccag cagattcttt tcttgttgat 2100gattttgaag gttattttgg ggatgacacg ttgttacatc gcaattattc tagcaatgga 2160gatccaatta cactatcgtt aacaagtgag tttaaaaata atggagaatt tggattgaag 2220tatgattatt cgattggctc gatgggttat gcagggaggc aaacatcact aggacctgtc 2280gattggagcg gagctaatgc ttttgaattt tggatgaaac atggacaact tgaagggaat 2340catttaactg tacaaattcg aataggtgat gttagctttg aaaaaaatct tgaattaatg 2400gatgctcatg aaggtgtagt gacaatcccg ttttctgaat ttgctccagc tgcttgggaa 2460aataagcctg gcgttatcat tgacgaacaa aaattgaaaa gagtgagtca atttgctctt 2520tacacaggcg gggctagaca atctggaaca atctactttg atgatttacg agcggtatat 2580gatgaaagtt taccatcagt tccagttccg aaagaggagg aagaggaaaa agaggtcgct 2640cctattattt atcattttga atctggaatt gataattggg aagggggaca agcaacacat 2700agcaatgggc acctcaaagt aacggttcgt ttaggtgaag gtcagcaaac cgaagtgaag 2760aaaacatcaa attataattt aacagggtat aattatatag tagctaatat aaaacatgac 2820gatacaggaa tgtttggtag tgacccgctt caagtgaaaa tctttacgaa agcaggaggt 2880tgggtatggg ctgattcagg aaatcaaccg atttactccg acgattatac tcaagttgtg 2940tatgatatta ctactttagc taacaaaaat gcagtccaag aaatcgggtt tgaatttttg 3000gctccttcag gttcttcagg gacgacgaat cctttcatag attcagtagc gattgttacg 3060agtctcgatc aattgtctga gcagccagag cagccagaac aaccaggaac accagatact 3120gatgataata aagaggataa agatagaaga aatgtagaag tgaacgagga aggacaaaaa 3180ctacccaaaa cagcaacgtc aatatttaat tatttgctaa ttggttttgt ttttgtaggg 3240attggattta gtctatttat ttataaaaga agaaaaacag tg 328261094PRTArtificial Sequencesynthetic Bsp Man4 sequence 6Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Gly Lys 20 25 30 Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys 35 40 45 Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln 50 55 60 Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr 65 70 75 80 Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala 85 90 95 Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp 100 105 110 Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys 115 120 125 Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile 130 135 140 Thr Leu Ser Met His Pro Asp Asn

Phe Val Thr Gly Gly Pro Tyr Gly 145 150 155 160 Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys 165 170 175 His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His 180 185 190 Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro 195 200 205 Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr 210 215 220 Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu 225 230 235 240 Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly 245 250 255 Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly 260 265 270 Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp 275 280 285 Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala 290 295 300 Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr 305 310 315 320 Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu 325 330 335 Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala 340 345 350 Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn 355 360 365 Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly Gly Asp 370 375 380 His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp Tyr Thr 385 390 395 400 Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly Lys Glu 405 410 415 Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser Pro Ile 420 425 430 Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala Lys Val 435 440 445 Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp Gly Ser Ser 450 455 460 Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr Thr Gly 465 470 475 480 Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp Val Ile 485 490 495 Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu Thr Ile Arg 500 505 510 Leu Phe Val Lys Ile Pro Glu Met Ser Leu Leu Thr Leu Thr Phe Asp 515 520 525 Asp Asp Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro Glu Asp Gly 530 535 540 Val Thr Ser Glu Ile Asp His Ala Ile Val Asp Gly Asp Gly Lys Leu 545 550 555 560 Met Phe Ser Val Gln Gly Met Ser Pro Thr Glu Thr Trp Gln Glu Leu 565 570 575 Lys Leu Glu Leu Thr Glu Leu Ser Asp Val Asn Ile Asp Ala Val Lys 580 585 590 Lys Met Lys Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu Glu Gly Ser 595 600 605 Val Gln Gly Ile Val Gln Leu Pro Pro Asp Trp Glu Thr Lys Tyr Gly 610 615 620 Met Asn Glu Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr Val Thr Val 625 630 635 640 Asn Gly Ser Asp Tyr Lys Arg Leu Glu Val Thr Val Ser Ile Asp Asn 645 650 655 Gln Gly Gly Ala Thr Gly Ile Ala Leu Ser Leu Val Gly Ser Gln Leu 660 665 670 Asp Leu Leu Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu Leu Asn Ser 675 680 685 Phe Glu Ala Pro Pro Ala Asp Ser Phe Leu Val Asp Asp Phe Glu Gly 690 695 700 Tyr Phe Gly Asp Asp Thr Leu Leu His Arg Asn Tyr Ser Ser Asn Gly 705 710 715 720 Asp Pro Ile Thr Leu Ser Leu Thr Ser Glu Phe Lys Asn Asn Gly Glu 725 730 735 Phe Gly Leu Lys Tyr Asp Tyr Ser Ile Gly Ser Met Gly Tyr Ala Gly 740 745 750 Arg Gln Thr Ser Leu Gly Pro Val Asp Trp Ser Gly Ala Asn Ala Phe 755 760 765 Glu Phe Trp Met Lys His Gly Gln Leu Glu Gly Asn His Leu Thr Val 770 775 780 Gln Ile Arg Ile Gly Asp Val Ser Phe Glu Lys Asn Leu Glu Leu Met 785 790 795 800 Asp Ala His Glu Gly Val Val Thr Ile Pro Phe Ser Glu Phe Ala Pro 805 810 815 Ala Ala Trp Glu Asn Lys Pro Gly Val Ile Ile Asp Glu Gln Lys Leu 820 825 830 Lys Arg Val Ser Gln Phe Ala Leu Tyr Thr Gly Gly Ala Arg Gln Ser 835 840 845 Gly Thr Ile Tyr Phe Asp Asp Leu Arg Ala Val Tyr Asp Glu Ser Leu 850 855 860 Pro Ser Val Pro Val Pro Lys Glu Glu Glu Glu Glu Lys Glu Val Ala 865 870 875 880 Pro Ile Ile Tyr His Phe Glu Ser Gly Ile Asp Asn Trp Glu Gly Gly 885 890 895 Gln Ala Thr His Ser Asn Gly His Leu Lys Val Thr Val Arg Leu Gly 900 905 910 Glu Gly Gln Gln Thr Glu Val Lys Lys Thr Ser Asn Tyr Asn Leu Thr 915 920 925 Gly Tyr Asn Tyr Ile Val Ala Asn Ile Lys His Asp Asp Thr Gly Met 930 935 940 Phe Gly Ser Asp Pro Leu Gln Val Lys Ile Phe Thr Lys Ala Gly Gly 945 950 955 960 Trp Val Trp Ala Asp Ser Gly Asn Gln Pro Ile Tyr Ser Asp Asp Tyr 965 970 975 Thr Gln Val Val Tyr Asp Ile Thr Thr Leu Ala Asn Lys Asn Ala Val 980 985 990 Gln Glu Ile Gly Phe Glu Phe Leu Ala Pro Ser Gly Ser Ser Gly Thr 995 1000 1005 Thr Asn Pro Phe Ile Asp Ser Val Ala Ile Val Thr Ser Leu Asp 1010 1015 1020 Gln Leu Ser Glu Gln Pro Glu Gln Pro Glu Gln Pro Gly Thr Pro 1025 1030 1035 Asp Thr Asp Asp Asn Lys Glu Asp Lys Asp Arg Arg Asn Val Glu 1040 1045 1050 Val Asn Glu Glu Gly Gln Lys Leu Pro Lys Thr Ala Thr Ser Ile 1055 1060 1065 Phe Asn Tyr Leu Leu Ile Gly Phe Val Phe Val Gly Ile Gly Phe 1070 1075 1080 Ser Leu Phe Ile Tyr Lys Arg Arg Lys Thr Val 1085 1090 71065PRTArtificial Sequencesynthetic Bsp Man4 sequence 7Ala Gly Lys Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp 1 5 10 15 Ala Ser Lys Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser 20 25 30 Gly Ser Gln Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu 35 40 45 Thr Leu Thr Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val 50 55 60 Phe Asn Ala Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn 65 70 75 80 Ser Leu Asp Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln 85 90 95 Ser Ile Lys Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly 100 105 110 Gly Ile Ile Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly 115 120 125 Pro Tyr Gly Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly 130 135 140 Gly Ser Lys His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala 145 150 155 160 Leu Ala His Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile 165 170 175 Phe Arg Pro Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala 180 185 190 Ser Thr Thr Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val 195 200 205 Glu Tyr Leu Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe 210 215 220 Ser Pro Gly Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr 225 230 235 240 Tyr Pro Gly Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp 245 250 255 Asn Lys Asp Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys 260 265 270 Asp Leu Ala Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala 275 280 285 Ala Phe Thr Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly 290 295 300 Asn Thr Leu Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp 305 310 315 320 Glu Asp Ala Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly 325 330 335 Trp Pro Asn Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu 340 345 350 Gly Gly Asp His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp 355 360 365 Asp Tyr Thr Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr 370 375 380 Gly Lys Glu Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile 385 390 395 400 Ser Pro Ile Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln 405 410 415 Ala Lys Val Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp 420 425 430 Gly Ser Ser Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr 435 440 445 Tyr Thr Gly Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val 450 455 460 Asp Val Ile Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu 465 470 475 480 Thr Ile Arg Leu Phe Val Lys Ile Pro Glu Met Ser Leu Leu Thr Leu 485 490 495 Thr Phe Asp Asp Asp Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro 500 505 510 Glu Asp Gly Val Thr Ser Glu Ile Asp His Ala Ile Val Asp Gly Asp 515 520 525 Gly Lys Leu Met Phe Ser Val Gln Gly Met Ser Pro Thr Glu Thr Trp 530 535 540 Gln Glu Leu Lys Leu Glu Leu Thr Glu Leu Ser Asp Val Asn Ile Asp 545 550 555 560 Ala Val Lys Lys Met Lys Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu 565 570 575 Glu Gly Ser Val Gln Gly Ile Val Gln Leu Pro Pro Asp Trp Glu Thr 580 585 590 Lys Tyr Gly Met Asn Glu Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr 595 600 605 Val Thr Val Asn Gly Ser Asp Tyr Lys Arg Leu Glu Val Thr Val Ser 610 615 620 Ile Asp Asn Gln Gly Gly Ala Thr Gly Ile Ala Leu Ser Leu Val Gly 625 630 635 640 Ser Gln Leu Asp Leu Leu Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu 645 650 655 Leu Asn Ser Phe Glu Ala Pro Pro Ala Asp Ser Phe Leu Val Asp Asp 660 665 670 Phe Glu Gly Tyr Phe Gly Asp Asp Thr Leu Leu His Arg Asn Tyr Ser 675 680 685 Ser Asn Gly Asp Pro Ile Thr Leu Ser Leu Thr Ser Glu Phe Lys Asn 690 695 700 Asn Gly Glu Phe Gly Leu Lys Tyr Asp Tyr Ser Ile Gly Ser Met Gly 705 710 715 720 Tyr Ala Gly Arg Gln Thr Ser Leu Gly Pro Val Asp Trp Ser Gly Ala 725 730 735 Asn Ala Phe Glu Phe Trp Met Lys His Gly Gln Leu Glu Gly Asn His 740 745 750 Leu Thr Val Gln Ile Arg Ile Gly Asp Val Ser Phe Glu Lys Asn Leu 755 760 765 Glu Leu Met Asp Ala His Glu Gly Val Val Thr Ile Pro Phe Ser Glu 770 775 780 Phe Ala Pro Ala Ala Trp Glu Asn Lys Pro Gly Val Ile Ile Asp Glu 785 790 795 800 Gln Lys Leu Lys Arg Val Ser Gln Phe Ala Leu Tyr Thr Gly Gly Ala 805 810 815 Arg Gln Ser Gly Thr Ile Tyr Phe Asp Asp Leu Arg Ala Val Tyr Asp 820 825 830 Glu Ser Leu Pro Ser Val Pro Val Pro Lys Glu Glu Glu Glu Glu Lys 835 840 845 Glu Val Ala Pro Ile Ile Tyr His Phe Glu Ser Gly Ile Asp Asn Trp 850 855 860 Glu Gly Gly Gln Ala Thr His Ser Asn Gly His Leu Lys Val Thr Val 865 870 875 880 Arg Leu Gly Glu Gly Gln Gln Thr Glu Val Lys Lys Thr Ser Asn Tyr 885 890 895 Asn Leu Thr Gly Tyr Asn Tyr Ile Val Ala Asn Ile Lys His Asp Asp 900 905 910 Thr Gly Met Phe Gly Ser Asp Pro Leu Gln Val Lys Ile Phe Thr Lys 915 920 925 Ala Gly Gly Trp Val Trp Ala Asp Ser Gly Asn Gln Pro Ile Tyr Ser 930 935 940 Asp Asp Tyr Thr Gln Val Val Tyr Asp Ile Thr Thr Leu Ala Asn Lys 945 950 955 960 Asn Ala Val Gln Glu Ile Gly Phe Glu Phe Leu Ala Pro Ser Gly Ser 965 970 975 Ser Gly Thr Thr Asn Pro Phe Ile Asp Ser Val Ala Ile Val Thr Ser 980 985 990 Leu Asp Gln Leu Ser Glu Gln Pro Glu Gln Pro Glu Gln Pro Gly Thr 995 1000 1005 Pro Asp Thr Asp Asp Asn Lys Glu Asp Lys Asp Arg Arg Asn Val 1010 1015 1020 Glu Val Asn Glu Glu Gly Gln Lys Leu Pro Lys Thr Ala Thr Ser 1025 1030 1035 Ile Phe Asn Tyr Leu Leu Ile Gly Phe Val Phe Val Gly Ile Gly 1040 1045 1050 Phe Ser Leu Phe Ile Tyr Lys Arg Arg Lys Thr Val 1055 1060 1065 81062PRTBacillus sp. SWT81 8Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys 1 5 10 15 Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln 20 25 30 Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr 35 40 45 Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala 50 55 60 Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp 65 70 75 80 Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys 85 90 95 Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile 100 105 110 Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly 115 120 125 Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys 130 135 140 His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His 145 150 155 160 Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro 165 170 175 Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr 180 185 190 Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu 195 200 205 Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly 210 215 220 Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly 225 230 235 240 Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp 245 250 255 Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala 260 265 270 Met Ile Ser Arg Leu Ala Glu Gln

Lys Glu Lys Val Ala Ala Phe Thr 275 280 285 Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu 290 295 300 Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala 305 310 315 320 Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn 325 330 335 Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly Gly Asp 340 345 350 His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp Tyr Thr 355 360 365 Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly Lys Glu 370 375 380 Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser Pro Ile 385 390 395 400 Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala Lys Val 405 410 415 Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp Gly Ser Ser 420 425 430 Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr Thr Gly 435 440 445 Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp Val Ile 450 455 460 Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu Thr Ile Arg 465 470 475 480 Leu Phe Val Lys Ile Pro Glu Met Ser Leu Leu Thr Leu Thr Phe Asp 485 490 495 Asp Asp Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro Glu Asp Gly 500 505 510 Val Thr Ser Glu Ile Asp His Ala Ile Val Asp Gly Asp Gly Lys Leu 515 520 525 Met Phe Ser Val Gln Gly Met Ser Pro Thr Glu Thr Trp Gln Glu Leu 530 535 540 Lys Leu Glu Leu Thr Glu Leu Ser Asp Val Asn Ile Asp Ala Val Lys 545 550 555 560 Lys Met Lys Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu Glu Gly Ser 565 570 575 Val Gln Gly Ile Val Gln Leu Pro Pro Asp Trp Glu Thr Lys Tyr Gly 580 585 590 Met Asn Glu Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr Val Thr Val 595 600 605 Asn Gly Ser Asp Tyr Lys Arg Leu Glu Val Thr Val Ser Ile Asp Asn 610 615 620 Gln Gly Gly Ala Thr Gly Ile Ala Leu Ser Leu Val Gly Ser Gln Leu 625 630 635 640 Asp Leu Leu Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu Leu Asn Ser 645 650 655 Phe Glu Ala Pro Pro Ala Asp Ser Phe Leu Val Asp Asp Phe Glu Gly 660 665 670 Tyr Phe Gly Asp Asp Thr Leu Leu His Arg Asn Tyr Ser Ser Asn Gly 675 680 685 Asp Pro Ile Thr Leu Ser Leu Thr Ser Glu Phe Lys Asn Asn Gly Glu 690 695 700 Phe Gly Leu Lys Tyr Asp Tyr Ser Ile Gly Ser Met Gly Tyr Ala Gly 705 710 715 720 Arg Gln Thr Ser Leu Gly Pro Val Asp Trp Ser Gly Ala Asn Ala Phe 725 730 735 Glu Phe Trp Met Lys His Gly Gln Leu Glu Gly Asn His Leu Thr Val 740 745 750 Gln Ile Arg Ile Gly Asp Val Ser Phe Glu Lys Asn Leu Glu Leu Met 755 760 765 Asp Ala His Glu Gly Val Val Thr Ile Pro Phe Ser Glu Phe Ala Pro 770 775 780 Ala Ala Trp Glu Asn Lys Pro Gly Val Ile Ile Asp Glu Gln Lys Leu 785 790 795 800 Lys Arg Val Ser Gln Phe Ala Leu Tyr Thr Gly Gly Ala Arg Gln Ser 805 810 815 Gly Thr Ile Tyr Phe Asp Asp Leu Arg Ala Val Tyr Asp Glu Ser Leu 820 825 830 Pro Ser Val Pro Val Pro Lys Glu Glu Glu Glu Glu Lys Glu Val Ala 835 840 845 Pro Ile Ile Tyr His Phe Glu Ser Gly Ile Asp Asn Trp Glu Gly Gly 850 855 860 Gln Ala Thr His Ser Asn Gly His Leu Lys Val Thr Val Arg Leu Gly 865 870 875 880 Glu Gly Gln Gln Thr Glu Val Lys Lys Thr Ser Asn Tyr Asn Leu Thr 885 890 895 Gly Tyr Asn Tyr Ile Val Ala Asn Ile Lys His Asp Asp Thr Gly Met 900 905 910 Phe Gly Ser Asp Pro Leu Gln Val Lys Ile Phe Thr Lys Ala Gly Gly 915 920 925 Trp Val Trp Ala Asp Ser Gly Asn Gln Pro Ile Tyr Ser Asp Asp Tyr 930 935 940 Thr Gln Val Val Tyr Asp Ile Thr Thr Leu Ala Asn Lys Asn Ala Val 945 950 955 960 Gln Glu Ile Gly Phe Glu Phe Leu Ala Pro Ser Gly Ser Ser Gly Thr 965 970 975 Thr Asn Pro Phe Ile Asp Ser Val Ala Ile Val Thr Ser Leu Asp Gln 980 985 990 Leu Ser Glu Gln Pro Glu Gln Pro Glu Gln Pro Gly Thr Pro Asp Thr 995 1000 1005 Asp Asp Asn Lys Glu Asp Lys Asp Arg Arg Asn Val Glu Val Asn 1010 1015 1020 Glu Glu Gly Gln Lys Leu Pro Lys Thr Ala Thr Ser Ile Phe Asn 1025 1030 1035 Tyr Leu Leu Ile Gly Phe Val Phe Val Gly Ile Gly Phe Ser Leu 1040 1045 1050 Phe Ile Tyr Lys Arg Arg Lys Thr Val 1055 1060 9296PRTBacillus sp. SWT81 9Asp Glu Asp Ala Ser Lys Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln 1 5 10 15 Asp Val Ser Gly Ser Gln Val Leu Phe Gly Gln Gln His Ala Thr Asp 20 25 30 Glu Gly Leu Thr Leu Thr Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln 35 40 45 Ser Glu Val Phe Asn Ala Val Gly Asp Tyr Pro Ala Val Phe Gly Trp 50 55 60 Asp Thr Asn Ser Leu Asp Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn 65 70 75 80 Val Glu Gln Ser Ile Lys Asn Thr Ala Gln Ser Met Lys Val Ala His 85 90 95 Asp Leu Gly Gly Ile Ile Thr Leu Ser Met His Pro Asp Asn Phe Val 100 105 110 Thr Gly Gly Pro Tyr Gly Asp Thr Thr Gly Asn Val Val Lys Glu Ile 115 120 125 Leu Pro Gly Gly Ser Lys His Ala Glu Phe Asn Ala Trp Leu Asp Asn 130 135 140 Ile Ala Ala Leu Ala His Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile 145 150 155 160 Pro Met Ile Phe Arg Pro Phe His Glu Gln Thr Gly Ser Trp Phe Trp 165 170 175 Trp Gly Ala Ser Thr Thr Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg 180 185 190 Tyr Thr Val Glu Tyr Leu Arg Asp Val Lys Gly Val Asn Asn Ile Leu 195 200 205 Tyr Gly Phe Ser Pro Gly Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr 210 215 220 Leu Glu Thr Tyr Pro Gly Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp 225 230 235 240 Asn Tyr Asp Asn Lys Asp Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly 245 250 255 Met Val Lys Asp Leu Ala Met Ile Ser Arg Leu Ala Glu Gln Lys Glu 260 265 270 Lys Val Ala Ala Phe Thr Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn 275 280 285 Arg Gln Gly Asn Thr Leu Asp Trp 290 295 10469PRTBacillus sp. SWT81 10Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys Tyr Thr Lys Glu 1 5 10 15 Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln Val Leu Phe Gly 20 25 30 Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr Asn Pro Ala Pro 35 40 45 Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala Val Gly Asp Tyr 50 55 60 Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp Gly Arg Glu Lys 65 70 75 80 Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys Asn Thr Ala Gln 85 90 95 Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile Thr Leu Ser Met 100 105 110 His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly Asp Thr Thr Gly 115 120 125 Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys His Ala Glu Phe 130 135 140 Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His Glu Leu Lys Asp 145 150 155 160 Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro Phe His Glu Gln 165 170 175 Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr Ser Pro Glu Gln 180 185 190 Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu Arg Asp Val Lys 195 200 205 Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly Ala Gly Pro Ala 210 215 220 Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly Asp Asp Tyr Val 225 230 235 240 Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp Asn Ala Gly Ser 245 250 255 Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala Met Ile Ser Arg 260 265 270 Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr Glu Tyr Gly Tyr 275 280 285 Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu Asp Trp Tyr Thr 290 295 300 Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala Arg Lys Ile Ser 305 310 315 320 Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn Asn Met Tyr Val 325 330 335 Pro Tyr Arg Asp Ile His Asn Glu Leu Gly Gly Asp His Glu Leu Leu 340 345 350 Pro Asp Phe Glu Ala Phe His Ala Asp Asp Tyr Thr Ala Phe Arg Asp 355 360 365 Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly Lys Glu Tyr Thr Val Ser 370 375 380 Pro His Glu Pro Phe Met Tyr Val Ile Ser Pro Ile Thr Gly Ser Thr 385 390 395 400 Val Thr Ser Glu Thr Val Thr Ile Gln Ala Lys Val Ala Asn Asp Glu 405 410 415 His Ala Arg Val Thr Phe Arg Val Asp Gly Ser Ser Leu Glu Glu Glu 420 425 430 Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr Thr Gly Ser Phe Thr Pro 435 440 445 Asp Ala Ala Val Asn Gly Gly Ala Val Asp Val Ile Val Ala Tyr Tyr 450 455 460 Ser Ser Gly Glu Lys 465 11383PRTBacillus sp. SWT81 11Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys Tyr Thr Lys Glu 1 5 10 15 Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln Val Leu Phe Gly 20 25 30 Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr Asn Pro Ala Pro 35 40 45 Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala Val Gly Asp Tyr 50 55 60 Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp Gly Arg Glu Lys 65 70 75 80 Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys Asn Thr Ala Gln 85 90 95 Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile Thr Leu Ser Met 100 105 110 His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly Asp Thr Thr Gly 115 120 125 Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys His Ala Glu Phe 130 135 140 Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His Glu Leu Lys Asp 145 150 155 160 Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro Phe His Glu Gln 165 170 175 Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr Ser Pro Glu Gln 180 185 190 Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu Arg Asp Val Lys 195 200 205 Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly Ala Gly Pro Ala 210 215 220 Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly Asp Asp Tyr Val 225 230 235 240 Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp Asn Ala Gly Ser 245 250 255 Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala Met Ile Ser Arg 260 265 270 Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr Glu Tyr Gly Tyr 275 280 285 Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu Asp Trp Tyr Thr 290 295 300 Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala Arg Lys Ile Ser 305 310 315 320 Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn Asn Met Tyr Val 325 330 335 Pro Tyr Arg Asp Ile His Asn Glu Leu Gly Gly Asp His Glu Leu Leu 340 345 350 Pro Asp Phe Glu Ala Phe His Ala Asp Asp Tyr Thr Ala Phe Arg Asp 355 360 365 Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly Lys Glu Tyr Thr Val 370 375 380 12849PRTArtificial Sequencesynthetic Bsp Man4 sequence 12Ala Gly Lys Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp 1 5 10 15 Ala Ser Lys Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser 20 25 30 Gly Ser Gln Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu 35 40 45 Thr Leu Thr Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val 50 55 60 Phe Asn Ala Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn 65 70 75 80 Ser Leu Asp Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln 85 90 95 Ser Ile Lys Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly 100 105 110 Gly Ile Ile Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly 115 120 125 Pro Tyr Gly Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly 130 135 140 Gly Ser Lys His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala 145 150 155 160 Leu Ala His Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile 165 170 175 Phe Arg Pro Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala 180 185 190 Ser Thr Thr Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val 195 200 205 Glu Tyr Leu Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe 210 215 220 Ser Pro Gly Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr 225 230 235 240 Tyr Pro Gly Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp 245 250 255 Asn Lys Asp Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys 260 265 270 Asp Leu Ala Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala 275 280 285 Ala Phe Thr Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly 290 295 300 Asn Thr Leu Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp 305 310 315 320 Glu Asp Ala Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly 325 330 335 Trp Pro Asn Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu 340 345 350

Gly Gly Asp His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp 355 360 365 Asp Tyr Thr Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr 370 375 380 Gly Lys Glu Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile 385 390 395 400 Ser Pro Ile Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln 405 410 415 Ala Lys Val Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp 420 425 430 Gly Ser Ser Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr 435 440 445 Tyr Thr Gly Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val 450 455 460 Asp Val Ile Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu 465 470 475 480 Thr Ile Arg Leu Phe Val Lys Ile Pro Glu Met Ser Leu Leu Thr Leu 485 490 495 Thr Phe Asp Asp Asp Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro 500 505 510 Glu Asp Gly Val Thr Ser Glu Ile Asp His Ala Ile Val Asp Gly Asp 515 520 525 Gly Lys Leu Met Phe Ser Val Gln Gly Met Ser Pro Thr Glu Thr Trp 530 535 540 Gln Glu Leu Lys Leu Glu Leu Thr Glu Leu Ser Asp Val Asn Ile Asp 545 550 555 560 Ala Val Lys Lys Met Lys Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu 565 570 575 Glu Gly Ser Val Gln Gly Ile Val Gln Leu Pro Pro Asp Trp Glu Thr 580 585 590 Lys Tyr Gly Met Asn Glu Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr 595 600 605 Val Thr Val Asn Gly Ser Asp Tyr Lys Arg Leu Glu Val Thr Val Ser 610 615 620 Ile Asp Asn Gln Gly Gly Ala Thr Gly Ile Ala Leu Ser Leu Val Gly 625 630 635 640 Ser Gln Leu Asp Leu Leu Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu 645 650 655 Leu Asn Ser Phe Glu Ala Pro Pro Ala Asp Ser Phe Leu Val Asp Asp 660 665 670 Phe Glu Gly Tyr Phe Gly Asp Asp Thr Leu Leu His Arg Asn Tyr Ser 675 680 685 Ser Asn Gly Asp Pro Ile Thr Leu Ser Leu Thr Ser Glu Phe Lys Asn 690 695 700 Asn Gly Glu Phe Gly Leu Lys Tyr Asp Tyr Ser Ile Gly Ser Met Gly 705 710 715 720 Tyr Ala Gly Arg Gln Thr Ser Leu Gly Pro Val Asp Trp Ser Gly Ala 725 730 735 Asn Ala Phe Glu Phe Trp Met Lys His Gly Gln Leu Glu Gly Asn His 740 745 750 Leu Thr Val Gln Ile Arg Ile Gly Asp Val Ser Phe Glu Lys Asn Leu 755 760 765 Glu Leu Met Asp Ala His Glu Gly Val Val Thr Ile Pro Phe Ser Glu 770 775 780 Phe Ala Pro Ala Ala Trp Glu Asn Lys Pro Gly Val Ile Ile Asp Glu 785 790 795 800 Gln Lys Leu Lys Arg Val Ser Gln Phe Ala Leu Tyr Thr Gly Gly Ala 805 810 815 Arg Gln Ser Gly Thr Ile Tyr Phe Asp Asp Leu Arg Ala Val Tyr Asp 820 825 830 Glu Ser Leu Pro Ser Val Pro Val Pro Lys Glu Glu Glu Glu Glu Lys 835 840 845 Glu 13669PRTArtificial Sequencesynthetic Bsp Man4 sequence 13Ala Gly Lys Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp 1 5 10 15 Ala Ser Lys Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser 20 25 30 Gly Ser Gln Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu 35 40 45 Thr Leu Thr Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val 50 55 60 Phe Asn Ala Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn 65 70 75 80 Ser Leu Asp Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln 85 90 95 Ser Ile Lys Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly 100 105 110 Gly Ile Ile Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly 115 120 125 Pro Tyr Gly Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly 130 135 140 Gly Ser Lys His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala 145 150 155 160 Leu Ala His Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile 165 170 175 Phe Arg Pro Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala 180 185 190 Ser Thr Thr Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val 195 200 205 Glu Tyr Leu Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe 210 215 220 Ser Pro Gly Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr 225 230 235 240 Tyr Pro Gly Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp 245 250 255 Asn Lys Asp Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys 260 265 270 Asp Leu Ala Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala 275 280 285 Ala Phe Thr Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly 290 295 300 Asn Thr Leu Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp 305 310 315 320 Glu Asp Ala Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly 325 330 335 Trp Pro Asn Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu 340 345 350 Gly Gly Asp His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp 355 360 365 Asp Tyr Thr Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr 370 375 380 Gly Lys Glu Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile 385 390 395 400 Ser Pro Ile Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln 405 410 415 Ala Lys Val Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp 420 425 430 Gly Ser Ser Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr 435 440 445 Tyr Thr Gly Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val 450 455 460 Asp Val Ile Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu 465 470 475 480 Thr Ile Arg Leu Phe Val Lys Ile Pro Glu Met Ser Leu Leu Thr Leu 485 490 495 Thr Phe Asp Asp Asp Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro 500 505 510 Glu Asp Gly Val Thr Ser Glu Ile Asp His Ala Ile Val Asp Gly Asp 515 520 525 Gly Lys Leu Met Phe Ser Val Gln Gly Met Ser Pro Thr Glu Thr Trp 530 535 540 Gln Glu Leu Lys Leu Glu Leu Thr Glu Leu Ser Asp Val Asn Ile Asp 545 550 555 560 Ala Val Lys Lys Met Lys Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu 565 570 575 Glu Gly Ser Val Gln Gly Ile Val Gln Leu Pro Pro Asp Trp Glu Thr 580 585 590 Lys Tyr Gly Met Asn Glu Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr 595 600 605 Val Thr Val Asn Gly Ser Asp Tyr Lys Arg Leu Glu Val Thr Val Ser 610 615 620 Ile Asp Asn Gln Gly Gly Ala Thr Gly Ile Ala Leu Ser Leu Val Gly 625 630 635 640 Ser Gln Leu Asp Leu Leu Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu 645 650 655 Leu Asn Ser Phe Glu Ala Pro Pro Ala Asp Ser Phe Leu 660 665 14492PRTArtificial Sequencesynthetic Bsp Man4 sequence 14Ala Gly Lys Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp 1 5 10 15 Ala Ser Lys Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser 20 25 30 Gly Ser Gln Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu 35 40 45 Thr Leu Thr Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val 50 55 60 Phe Asn Ala Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn 65 70 75 80 Ser Leu Asp Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln 85 90 95 Ser Ile Lys Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly 100 105 110 Gly Ile Ile Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly 115 120 125 Pro Tyr Gly Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly 130 135 140 Gly Ser Lys His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala 145 150 155 160 Leu Ala His Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile 165 170 175 Phe Arg Pro Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala 180 185 190 Ser Thr Thr Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val 195 200 205 Glu Tyr Leu Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe 210 215 220 Ser Pro Gly Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr 225 230 235 240 Tyr Pro Gly Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp 245 250 255 Asn Lys Asp Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys 260 265 270 Asp Leu Ala Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala 275 280 285 Ala Phe Thr Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly 290 295 300 Asn Thr Leu Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp 305 310 315 320 Glu Asp Ala Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly 325 330 335 Trp Pro Asn Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu 340 345 350 Gly Gly Asp His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp 355 360 365 Asp Tyr Thr Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr 370 375 380 Gly Lys Glu Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile 385 390 395 400 Ser Pro Ile Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln 405 410 415 Ala Lys Val Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp 420 425 430 Gly Ser Ser Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr 435 440 445 Tyr Thr Gly Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val 450 455 460 Asp Val Ile Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu 465 470 475 480 Thr Ile Arg Leu Phe Val Lys Ile Pro Glu Met Ser 485 490 15556PRTBacillus halodurans 15Ala Ser Gly Gln Glu Leu Lys Met Thr Asp Gln Asn Ala Ser Gln Tyr 1 5 10 15 Thr Lys Glu Leu Phe Ala Phe Leu Arg Asp Val Ser Gly Lys Gln Val 20 25 30 Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Arg Gly 35 40 45 Thr Gly Asn Arg Ile Gly Ser Thr Glu Ser Glu Val Lys Asn Ala Val 50 55 60 Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp Gly 65 70 75 80 Arg Glu Lys Pro Gly Asn Asp Glu Pro Ser Gln Glu Gln Arg Ile Leu 85 90 95 Asn Thr Ala Ala Ser Met Lys Ala Ala His Asp Leu Gly Gly Ile Ile 100 105 110 Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Ala Tyr Gly 115 120 125 Asp Thr Thr Gly Asn Val Val Gln Glu Ile Leu Pro Gly Gly Ser Lys 130 135 140 His Glu Glu Phe Asn Ala Trp Leu Asp Asn Leu Ala Ala Leu Ala His 145 150 155 160 Glu Leu Lys Asp Asp Asn Gly Lys His Ile Pro Ile Ile Phe Arg Pro 165 170 175 Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr 180 185 190 Thr Pro Glu Gln Tyr Lys Ala Ile Tyr Arg Tyr Thr Val Glu Tyr Leu 195 200 205 Arg Asp Val Lys Gly Ala Asn Asn Phe Leu Tyr Gly Phe Ser Pro Gly 210 215 220 Ala Gly Pro Ala Gly Asp Leu Asn Arg Tyr Met Glu Thr Tyr Pro Gly 225 230 235 240 Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Ser 245 250 255 Asn Ala Gly Ser Glu Ala Trp Ile Gln Gly Val Val Thr Asp Leu Ala 260 265 270 Met Leu Val Asp Leu Ala Glu Glu Lys Gly Lys Ile Ala Ala Phe Thr 275 280 285 Glu Tyr Gly Tyr Ser Ala Thr Gly Met Asn Arg Thr Gly Asn Thr Leu 290 295 300 Asp Trp Tyr Thr Arg Leu Leu Asn Ala Ile Lys Glu Asp Pro Lys Ala 305 310 315 320 Ser Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Phe Pro Asn 325 330 335 Asn Met Tyr Val Pro Tyr Lys Asp Ile His Gly Asp Leu Gly Gly Asp 340 345 350 His Glu Leu Leu Pro Asp Phe Ile Lys Phe Phe Glu Asp Asp Tyr Ser 355 360 365 Ala Phe Thr Gly Asp Ile Lys Gly Asn Val Tyr Asp Thr Gly Ile Glu 370 375 380 Tyr Thr Val Ala Pro His Glu Arg Leu Met Tyr Val Leu Ser Pro Ile 385 390 395 400 Thr Gly Thr Thr Ile Thr Asp Thr Val Thr Leu Arg Ala Lys Val Leu 405 410 415 Asn Asp Asp Asn Ala Val Val Thr Tyr Arg Val Glu Gly Ser Asp Val 420 425 430 Glu His Glu Met Thr Leu Ala Asp Ser Gly Tyr Tyr Thr Ala Lys Tyr 435 440 445 Ser Pro Thr Ala Glu Val Asn Gly Gly Ser Val Asp Leu Thr Val Thr 450 455 460 Tyr Trp Ser Gly Glu Glu Lys Val Gln Asp Glu Val Ile Arg Leu Tyr 465 470 475 480 Val Lys Ala Ser Glu Ile Ser Leu Tyr Lys Leu Thr Phe Asp Glu Asp 485 490 495 Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro Glu Asp Gly Ile Thr 500 505 510 Ser Asp Val Ser His Val Ser Phe Asp Gly Asn Gly Lys Leu Lys Phe 515 520 525 Ala Val Asn Gly Met Ser Ser Glu Glu Trp Trp Gln Glu Leu Lys Leu 530 535 540 Glu Leu Thr Asp Leu Ser Asp Val Asn Leu Ala Lys 545 550 555 161066PRTBacillus cellulosilyticus 16Glu Glu Thr Arg Val Leu Lys Met Ser Asn Pro Asp Ala Ser Lys Tyr 1 5 10 15 Thr Lys Glu Leu Phe Ala Tyr Leu Gln Asp Val Gly Ser Asp Asn Val 20 25 30 Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Ile Lys Val 35 40 45 Glu Asn Gly Asp Ser Asn Phe Val Gly Ser Thr Gln Ser Glu Val Lys 50 55 60 Asn Ala Val Gly Asp Tyr Pro Ala

Val Phe Gly Trp Asp Thr Asn Ser 65 70 75 80 Leu Asp Gly Arg Glu Arg Pro Gly Asn Pro Ile Ser Gly Glu Pro Leu 85 90 95 Thr Gln Glu Gln Arg Thr Gln Asn Leu Ala Lys Ser Met Ile Thr Ala 100 105 110 His Glu Leu Gly Gly Ile Ile Thr Leu Ser Met His Pro Asp Asn Phe 115 120 125 Val Thr Gly Glu Tyr Tyr Gly Asp Thr Asp Gly Asn Val Val Lys Thr 130 135 140 Ile Leu Pro Gly Gly Val His His Asp Asp Tyr Asn Glu Trp Leu Asp 145 150 155 160 Asn Ile Val Asp Leu Ser His Leu Val Val Asp Glu Asp Gly His His 165 170 175 Ile Pro Ile Ile Phe Arg Pro Phe His Glu Gln Asn Gly Ser Trp Phe 180 185 190 Trp Trp Gly Ala Ser Thr Thr Thr Pro Glu Gln Tyr Lys Ala Ile Phe 195 200 205 Arg Tyr Thr Val Glu Tyr Leu Arg Glu His Gly Ala Asn Asn Phe Leu 210 215 220 Ile Gly Phe Ser Pro Asn Gly Ala Ser Ala Gly Asp Leu Glu Gln Tyr 225 230 235 240 Leu Glu Thr Tyr Pro Gly Asp Asp Tyr Val Asp Ile Leu Gly Ile Asp 245 250 255 Arg Tyr Asp Thr Lys Ser Asn Ala Gly Ser Gln Glu Trp Leu Thr Ala 260 265 270 Val Ala Lys Asp Leu Ala Met Ile Ser Lys Glu Ala Glu Asp Arg Gly 275 280 285 Lys Ile Ser Ala Phe Thr Glu Phe Gly Tyr Ser Pro Thr Gly Met Asn 290 295 300 Glu Glu Gly Asn Asn Leu Gln Trp Trp Glu Asp Leu Leu Ser Ala Ile 305 310 315 320 Met Asp Asn Pro Asp Tyr Pro Glu Ala Ala Asn Ile Ala Phe Met Ser 325 330 335 Thr Trp Ala Asn Phe Gly Phe Pro Asn Asn Met Tyr Val Pro Tyr Arg 340 345 350 Asp Ile His Gly Asp Leu Gly Gly Asp His Glu Leu Leu Pro Thr Phe 355 360 365 Glu Glu Phe Tyr Asn Asp Glu Ser Thr Leu Phe Ser Glu Glu Val Lys 370 375 380 Gly Gln Ile Tyr His Ser Gly Lys Thr Leu Glu Thr Ala Glu His Asp 385 390 395 400 Ser Lys Met Tyr Thr Leu Ser Pro Thr Asp Gly Asp Thr Ile Thr Glu 405 410 415 Asn Lys Val Thr Leu Leu Thr Arg Val Val Asn Asp Asp Asp Ala Thr 420 425 430 Val Thr Tyr Ser Val Asp Gly Ser Glu Glu Val Glu Met Glu Leu Ala 435 440 445 Gly Arg Tyr Tyr Thr Ala Asp Trp Ile Pro Asn Ala Leu Gln Asn Gly 450 455 460 Gly Thr Ala Asn Ile Thr Ile Arg His Tyr Asp Gly Asn Asn Asn Glu 465 470 475 480 Val Ser Lys Glu Val Ile Arg Thr Tyr Leu Arg Val Pro Glu Ile Leu 485 490 495 Val Glu Glu Ile Thr Phe Asp Asp Ser Ile Glu Gly Ala Leu Asn Lys 500 505 510 Gly Thr Trp Pro Glu Val Gly Val Glu Phe Glu Leu Ser His Glu Lys 515 520 525 Leu Gly Gly Asp Gly Lys Leu Ala Leu Ser Val Ser Gly Met Pro Glu 530 535 540 Asp Glu Trp Trp Gln Glu Leu Lys Ile Gly Phe Glu Asp Leu Ser His 545 550 555 560 Val Asn Phe Asp Val Val Asn Gln Val Lys Phe Asp Val Leu Leu Pro 565 570 575 Glu Thr Val Ala Asp Gly Ala Ile Leu Ser Thr Val Leu Ala Ala Asp 580 585 590 Gly Asn Thr Lys Tyr Gly Glu Gly Thr Thr Glu Arg Asn Val Thr Asp 595 600 605 Leu Glu Ile Ile Glu Ile Asp Gly Val Glu Tyr Lys Leu Tyr Glu Thr 610 615 620 Thr Ile Asn Leu Glu Glu Ser Ile Thr Glu Gly Thr Glu Leu Gly Ile 625 630 635 640 Ile Gly Lys Gln Leu Asp Phe Ser Asn Lys Leu Tyr Leu Asp Asn Val 645 650 655 Arg Phe Leu Asn Ala Tyr Leu Glu Ala Pro Thr Asp Pro Leu Leu Val 660 665 670 Asp Asp Phe Glu Gly Tyr Leu Gly Asp Asn Asp Leu Leu Asn Arg Asn 675 680 685 Tyr Ser Asn Pro Gly Asp Arg Ile Leu Ile Ser Leu Ser Ser Glu His 690 695 700 Lys His Ser Gly Glu Tyr Gly Leu Gln Tyr Asp Trp Thr Ile Gly Ser 705 710 715 720 Ser Gly Tyr Ala Gly Arg Gln Thr Ser Leu Gly Pro Val Asp Trp Ser 725 730 735 Gly Thr Asn Ala Phe Gln Phe Trp Leu Lys His Asp Asp Leu Pro Asp 740 745 750 Asn Ser Leu Thr Val Gln Ile Gln Met Gly Gly Val Ser Phe Glu Ala 755 760 765 Ser Thr Asp Leu Asp Glu Ser Phe Glu Gly Ile Val Thr Ile Pro Phe 770 775 780 Val Asp Phe Ala Pro Ala His Trp Glu Gly Asn Gln Thr Ala Ile Ile 785 790 795 800 Asp Lys Pro Arg Leu Glu Arg Val Ser Gln Phe Ala Leu Tyr Met Gly 805 810 815 Gly Asn Glu Gly Ser Gly Thr Leu Tyr Phe Asp Asp Leu Arg Ala Val 820 825 830 Tyr Asp Glu Asp Ala Pro Pro Val Pro Glu Arg Glu Asp Ala Gly Glu 835 840 845 Ile Glu Pro Ile Ile Tyr Asp Phe Glu Ser Asp Leu Asp Gly Trp Gly 850 855 860 Thr Asn Met Ser Leu Ile Lys Asp Gly Asn Leu Val His Pro Val Gly 865 870 875 880 Leu Gly Glu Gly Asn Lys Thr Glu Ile Ala Lys Thr Ser Gly Tyr Asp 885 890 895 Leu Ser Gly His Asn Tyr Ile Val Ala Thr Val Lys His Asp Glu Glu 900 905 910 Gly Thr Phe Gly Asp Asp Pro Leu Asn Ala Lys Leu Phe Ile Lys Thr 915 920 925 Gly Ser Ala Trp Thr Trp Ala Asp Ser Gly Asp Phe Ser Leu Asn Ser 930 935 940 Asp Lys Tyr Val Glu Ile Val Phe Asp Ile Ser Asp Asn Ala Ala Arg 945 950 955 960 Glu Asn Val Gln Glu Ile Gly Leu Glu Phe Thr Ala Pro Ala Gly Ser 965 970 975 Asp Gly Thr Ser Asn Ala Tyr Ile Glu Ser Ile Lys Ile Leu Thr Ala 980 985 990 Leu Glu Glu Leu Pro Asp Thr Glu Asp Pro Gly Glu Thr Asp Glu Val 995 1000 1005 Gln Glu Leu Lys Asp Leu Ile Ser Asp Leu Lys Glu Arg Ile Lys 1010 1015 1020 Glu Leu Glu Asn Asn Thr Asp Val Glu Asp Phe Asp Lys Arg Val 1025 1030 1035 Gln Glu Leu Thr Asn Glu Leu Asn His Leu Lys Ala Lys Tyr Asn 1040 1045 1050 Asp Met Glu Lys Leu Val Pro Val Ile Glu Gln Arg Leu 1055 1060 1065 17510PRTCellulomonas fimi 17Met Pro Ser Ala Gln Ala Gln Glu Gln Ile Ile Asn Leu Val Asp Ala 1 5 10 15 Glu Ala Ser Thr Ser Thr Lys Gln Leu Phe Ser Tyr Leu Gln Ser Ile 20 25 30 Ser Gly Glu Lys Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly 35 40 45 Ile Thr Val Thr Gly Pro Gly Leu Arg Thr Gly Ser Thr Glu Ser Glu 50 55 60 Val Lys Asn Ser Val Gly Asp Tyr Pro Ala Leu Phe Gly Trp Asp Thr 65 70 75 80 Leu Ser Leu Asp Gly Tyr Glu Lys Pro Gly Ser Arg Glu Gln Ser Ala 85 90 95 Ala Glu Asn Arg Ala Asn Leu Ile Lys Ser Met Lys Thr Ala His Glu 100 105 110 Leu Gly Gly Ile Leu Thr Leu Ser Thr His Pro His Asn Phe Val Thr 115 120 125 Gly Gly Asp Phe Tyr Asp Thr Ser Gly Arg Val Val Lys Asn Ile Leu 130 135 140 Pro Gly Gly Ser Tyr Asn Ala Arg Phe Asn Glu Trp Leu Asp Asn Ile 145 150 155 160 Ala Ala Phe Ala Asn Asp Leu Lys Asp Asn Glu Gly Lys Asp Ile Pro 165 170 175 Val Ile Phe Arg Pro Phe His Glu Gln Thr Gly Gly Trp Phe Trp Trp 180 185 190 Gly Ala Gln Thr Thr Ser Ala Ala Glu Tyr Lys Glu Leu Tyr Arg Tyr 195 200 205 Thr Val Glu Tyr Leu Arg Asp Val Lys Gly Val Asp Asn Phe Leu Tyr 210 215 220 Ala Phe Ser Pro Gly Ala Ser Phe Asn Gly Asp Glu Glu Lys Tyr Leu 225 230 235 240 Lys Thr Tyr Pro Gly Asp Asp Tyr Val Asp Val Leu Gly Phe Asp Gln 245 250 255 Tyr Asp Asn Pro Asn Asn Pro Gly Ser Glu Gly Phe Leu Asn Thr Leu 260 265 270 Val Val Asp Leu Gly Met Leu Ser Lys Leu Ala Asp Ser Lys Gly Lys 275 280 285 Ile Ala Ala Leu Thr Glu Tyr Gly Leu Gly Leu Lys Thr Asn Gly Asn 290 295 300 Leu Asp Thr Gln Trp Phe Thr Arg Val Leu Asp Ala Ile Lys Ala Asp 305 310 315 320 Pro Tyr Ala Arg Lys Ile Ser Tyr Met Gln Thr Trp Ala Asn Phe Gly 325 330 335 Leu Asn Gly Asn Leu Phe Val Pro Tyr Lys Asn Ala Pro Asn Gly Leu 340 345 350 Gly Asp His Glu Leu Leu Pro Asp Phe Ile Asn Phe Tyr Lys Asp Pro 355 360 365 Tyr Ser Ala Phe Ser Lys Asp Val Gly Asn Ile Tyr Arg Gly Ala Val 370 375 380 Pro Glu Thr Val Ala Glu Lys Pro Phe Met His Ile Val Ser Pro Ile 385 390 395 400 Asp Arg Ser Leu Ser Leu Gln Lys Val Thr Pro Ile Ser Val Ser Val 405 410 415 Ile Gln Gly Lys Pro Lys Asp Ile Tyr Tyr Thr Val Asn Asp Lys Ala 420 425 430 Lys Lys Tyr Pro Leu Val Lys Gly Asp Gly Tyr Tyr Tyr Glu Gly Ser 435 440 445 Ala Ala Leu Lys Gly Asp Lys Ala Thr Ile His Val Thr Ala Glu Phe 450 455 460 Ala Asp Gly Thr Ser Gln Lys Gln Thr Ile Lys Val Tyr Leu Lys Glu 465 470 475 480 Pro Glu Lys Gln Pro Pro Thr Val Val Asp Thr Phe Glu Thr Tyr Tyr 485 490 495 Gly Asp Asp Glu Gln Leu Gln Ala Ala Phe Ala Thr Gln Gly 500 505 510 18964PRTBacillus sp. 18Ser Glu Gly Gln Ser Phe Lys Leu Val Asp Ser Asn Ala Ser Thr Leu 1 5 10 15 Thr Lys Ser Leu Tyr Ala Tyr Leu Gln Asp Thr Ser Gly Arg Gln Ile 20 25 30 Leu Phe Gly His Gln His Ala Val Asp Glu Gly Leu Thr Leu Thr Asn 35 40 45 Ser Gly Asp Arg Val Gly Ser Thr Gln Ser Glu Val Lys Asn Ala Val 50 55 60 Gly Asp Tyr Pro Ala Ile Phe Gly Trp Asp Thr Leu Ser Leu Asp Gly 65 70 75 80 Tyr Glu Lys Pro Gly Asn Glu Lys Asn Ser Gln Ala Gln Asn Arg Ala 85 90 95 Asn Val Val Gln Ser Met Arg Thr Val His Glu Leu Gly Gly Ile Ile 100 105 110 Ala Leu Ser Met His Pro Glu Asn Phe Val Thr Gly Asn Gln Tyr Asn 115 120 125 Asp Thr Ser Gly Asp Val Val Lys Asn Ile Leu Pro Asp Gly Ser His 130 135 140 His Glu Val Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Phe Ala His 145 150 155 160 Glu Leu Thr Asp Gln Ser Thr Gly Glu Leu Ile Pro Val Ile Phe Arg 165 170 175 Pro Phe His Glu Gln Asn Gly Gly Trp Phe Trp Trp Gly Ala Gln Thr 180 185 190 Thr Thr Ala Ser Glu Tyr Lys Ala Leu Tyr Arg Tyr Thr Val Asp Tyr 195 200 205 Leu Arg Asp Val Lys Gly Val Asn Asn Phe Leu Tyr Ala Phe Ser Pro 210 215 220 Asn Ala Pro Phe Asp Gly Asn Leu Thr Gln Tyr Leu Arg Thr Tyr Pro 225 230 235 240 Gly Asp Gln Tyr Val Asp Ile Phe Gly Leu Asp Gln Tyr Asp Asn Lys 245 250 255 Ala Asn Ala Gly Gln Ala Thr Phe Leu Asn Gly Leu Thr Gln Asp Leu 260 265 270 Ala Met Ile Ser Lys Leu Ala Asp Glu Lys Gly Lys Ile Ala Ala Phe 275 280 285 Thr Glu Tyr Gly Tyr Ser Pro Gln Gly Phe Asn Glu Thr Gly Asn Tyr 290 295 300 Leu Gln Trp Tyr Thr Ala Val Leu Glu Ala Ile Lys Lys Asp Pro Asn 305 310 315 320 Ala Ser Arg Ile Ala Tyr Met Gln Thr Trp Ala Asn Phe Gly Tyr Pro 325 330 335 Thr Asn Met Phe Val Pro Tyr Arg Asp Val Asn Gly Asn Leu Gly Gly 340 345 350 Asp His Glu Leu Leu Pro Asn Phe Val Glu Phe Tyr Glu Asp Asp Tyr 355 360 365 Ala Ala Phe Leu Thr Glu Ala Ser Gly Trp Asn Leu Tyr Gln Asp Ile 370 375 380 Ser Thr Ile Glu Gln Glu Pro Phe Met His Ile Val Thr Pro Thr Ala 385 390 395 400 Asn Ser Gln Ile Ser Glu Ala Val Thr Ile Arg Ala Arg Val Leu His 405 410 415 Asp Gln Pro Ser His Val Val Phe Glu Val Asn Asp Ser Gly Glu Glu 420 425 430 Ile Pro Met Ser Leu Asp Glu Asp Gly Phe Phe Tyr Met Gly Lys Trp 435 440 445 Thr Pro Asp Ala Ala Val Asn His Thr Thr Val Asn Ile Thr Val Arg 450 455 460 Ala Tyr Gly Glu Asn Gln Val Gln Glu Glu Thr Phe Pro Leu Val Val 465 470 475 480 Arg Val Ser Glu Met Leu Leu Lys Glu Tyr Thr Phe Asp Glu Gly Ile 485 490 495 Glu Gly Ile Gln Asn Asn Gly Thr Tyr Pro Asp Thr Ile Glu Thr Ser 500 505 510 Phe Glu His Gln Val Leu Asn Gly Asp Gly Lys Leu Lys Ile Asn Val 515 520 525 Ala Gly Leu Gln Ala Ser Asp Thr Trp Gln Glu Leu Lys Leu Glu Leu 530 535 540 Thr Asn Leu His Asp Val Gln Leu Gly Asn Val Asn Arg Val Lys Val 545 550 555 560 Asp Val Phe Ile Pro Lys Ala Ala Val Asn Gln Ser Ala Thr Ile Arg 565 570 575 Gly Ile Val Gln Leu Pro Pro Asp Trp Asp Thr Lys Tyr Gly Met Thr 580 585 590 Thr Thr Glu Lys Asn Leu Ser Asp Leu Gln Ser Val Val Ile Asp Glu 595 600 605 Glu Glu Tyr Val Glu Gly Gln Ile Thr Ile Asp Leu Thr Ser Pro Glu 610 615 620 Ala Ser Ala Ala Ala Thr Gly Leu Ala Leu Ser Leu Val Gly Asn Ala 625 630 635 640 Ile Asp Phe Thr Gly Ala Ile Tyr Val Asp Asn Ile Gln Leu Ile Gly 645 650 655 Val Ser Glu Glu Glu Val Ser Asp Pro Ala Ile Val Asp Asp Phe Glu 660 665 670 Ser Tyr Val Gly Asn Asp Asp Leu Leu Arg Asn Ala Trp Val Ala Ala 675 680 685 Asn Gly Gly Ile Ala Ile Ser Leu Asp Gln Glu Glu Lys Ser Ala Gly 690 695 700 Asp Tyr Gly Leu Ala Tyr Glu Tyr Ser Leu Ala Gly Ala Gly Ser Tyr 705 710 715 720 Thr Gly Ile Thr Lys Met Leu Gly Asn Arg Asp Trp Ser Ser Tyr Asn 725 730 735 Ser Leu Gln Phe Trp Met Asn Ser Asp Gly Asn Gly Gln Lys Leu Val 740 745 750 Ile Gln Ala Glu Ile Gly Gly Val His Phe Glu Ala Tyr Pro Ser Leu 755 760 765 Glu Ala Asn Glu Glu Gly Leu Val Thr Ile Gly Phe Asn Glu Phe Thr 770 775 780 Pro Ala Pro Trp Glu Ser Ala Ser Asn Leu Glu Lys

Leu Val Thr Glu 785 790 795 800 Glu Ala Leu Lys Asn Val Thr Lys Leu Ser Leu Tyr Ile Asn Ala Gln 805 810 815 Asp Glu Leu Asp Ser Ala Leu Val Ser Thr Leu Phe Phe Asp Glu Ile 820 825 830 Arg Ala Ala Tyr Val Glu Glu Glu Pro Gly Glu Glu Gly Glu Pro Gly 835 840 845 Glu Glu Gly Lys Ser Gly Glu Glu Gly Lys Pro Gly Glu Glu Gly Glu 850 855 860 Pro Gly Glu Glu Gly Glu Pro Gly Glu Glu Gly Lys Pro Gly Glu Glu 865 870 875 880 Gly Glu Leu Gly Glu Glu Gly Lys Pro Gly Glu Glu Gly Glu Leu Gly 885 890 895 Glu Glu Glu Glu Pro Gly Glu Glu Gly Glu Leu Gly Glu Glu Leu Glu 900 905 910 Val Gly His Lys Glu Gln Gly Asn Gln Ser Ser Ser Gly Ala Asn Lys 915 920 925 Leu Pro Ser Thr Ala Thr Asn Val Phe Asn Phe Leu Leu Ile Gly Thr 930 935 940 Leu Leu Val Ile Gly Ser Thr Ser Leu Leu Tyr Met Arg Arg Lys Lys 945 950 955 960 Ile Asn Asn Glu 19829PRTEnterococcus faecium 19Ser Tyr Ala Asp Ser Tyr Asn Met Val Asp Glu Arg Ala Ser Glu Lys 1 5 10 15 Thr Arg Gln Leu Phe Gly Phe Leu Gln Ala Thr Gln Asp Ser Ser Gly 20 25 30 Ile Met Phe Gly His Gln His Ala Leu Asp Glu Gly Val Thr Leu Thr 35 40 45 Gly Glu Ala Pro Arg Thr Gly Ser Thr Asp Ser Glu Val Lys Asn Ala 50 55 60 Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Gly Ser Leu Asp 65 70 75 80 Gly Gly Glu Lys Pro Gly Val Ala Gly Asp Val Glu Gln Ser Ile Gln 85 90 95 Asn Thr Ala Ile Ser Met Lys Thr Ala Tyr Asp Leu Gly Gly Val Ile 100 105 110 Val Leu Ser Met His Pro Arg Asn Phe Val Thr Gly Gly Ala Tyr Asn 115 120 125 Asp Leu Thr Gly Asn Val Val Gln Asn Ile Leu Pro Gly Gly Asp Tyr 130 135 140 Asn Asp Thr Phe Asn Ala Trp Leu Asp Gln Ile Ala Thr Leu Ser Tyr 145 150 155 160 Leu Leu Lys Asp Asp Asp Gly Asn Ser Ile Pro Phe Ile Phe Arg Pro 165 170 175 Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Glu Ser Thr Thr 180 185 190 Thr Thr Glu Gln Tyr Lys Ala Ile Tyr Arg Tyr Thr Val Asp Tyr Leu 195 200 205 Lys Asn Thr Lys Asp Val His Asn Ile Leu Tyr Ala Tyr Thr Pro Asn 210 215 220 Lys Met Thr Pro Gly Asp Glu Glu Arg Tyr Met Arg Thr Tyr Pro Gly 225 230 235 240 Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Ile Tyr Asp Gln Gln Glu 245 250 255 Asn Ala Gly Ser Glu Glu Phe Leu Asp Ser Val Val Gln Asp Leu Ser 260 265 270 Met Ile Thr Ser Ile Ala Glu Ser Lys Asn Lys Ile Ala Ala Leu Ser 275 280 285 Glu Phe Gly Tyr Ser Ala Val Gly Leu Lys Glu Thr Gly Asn Thr Leu 290 295 300 Asp Trp Tyr Thr Arg Leu Phe Asn Ala Ile Lys Asn Asn Thr Gln Ala 305 310 315 320 Ser Lys Ile Ala Tyr Met Leu Thr Trp Ala Asn Phe Gly Leu Pro Thr 325 330 335 Asn Leu Tyr Val Pro Tyr Arg Asp Val Asn Gly Asp Leu Gly Gly Asp 340 345 350 His Glu Met Leu Pro Asp Phe Val Ser Tyr Tyr Asp Asp Pro Ala Ser 355 360 365 Leu Phe Leu Glu Glu Val Lys Gly Asn Ile Tyr Ile Pro Glu Asn Ser 370 375 380 Gly Glu Thr Val Gln Gln Ser Ala Gln Phe Phe Val Leu Asn Pro Thr 385 390 395 400 Asn Lys Leu Thr Ile Thr Asn Ser Glu Leu Pro Ile Tyr Thr Met Ala 405 410 415 Thr Asn Asp Asp Ser Ala Glu Val Thr Tyr Glu Ile Glu Gly Ile Thr 420 425 430 Glu Glu Thr Ala Leu Thr Arg Gln Gly Asn Leu Phe Gly Gly Ser Phe 435 440 445 Asp Leu Gly Asp Asn Phe Lys Asn Gly Ser Leu Asn Leu Val Leu Arg 450 455 460 Tyr Tyr Ser Ala Gly Val Glu Val Glu Ser Glu Asn Ile Gln Val Tyr 465 470 475 480 Met Gln Ala Glu Ile Asn Glu Ser Glu Leu Leu Ile Asp Asp Phe Glu 485 490 495 Ser Tyr Leu Gly Glu Asp Glu Leu Leu Asn Gln Lys Tyr Val Ser Ser 500 505 510 Gly Asp Pro Ile Thr Met Ser Leu Ser Glu Val Lys Asn Ser Gly Asp 515 520 525 Tyr Gly Leu Lys Phe Asp Tyr Thr Ile Ala Ser Gln Gly Tyr Ser Gly 530 535 540 Arg Gln Leu Ser Val Glu Lys Asp Trp Ser Asn Ala Thr Gly Ile Ser 545 550 555 560 Phe Trp Met Ala Asn Glu Val Ala Ser Gln Asp Thr Leu Thr Ile Gln 565 570 575 Ile Arg Ile Gly Ser Val Ser Phe Glu Ala Tyr Val Asp Leu Ser Ser 580 585 590 Pro Tyr Thr Gly Ile Val Glu Ile Pro Phe Asp Glu Phe Val Pro Ala 595 600 605 Ser Trp Glu Gln Asn Gln Ser Ala Glu Ile Asn Ser Glu Thr Leu Gln 610 615 620 Ser Val Ser Leu Phe Ala Leu Tyr Met Gly Gly Ser Lys Gly Glu Gly 625 630 635 640 Thr Leu Tyr Phe Asp Asp Ile Gln Ala Val Asp Ala Ser Ser Val Val 645 650 655 Asp Pro Thr Glu Tyr Thr Val Thr Val Ile His Glu Asp Val Asp Gly 660 665 670 Asn Ile Leu Leu Thr Glu Asp Ile Gln Ala Glu Glu Gly Lys Thr Val 675 680 685 Thr Val Glu Ser Lys Gln Phe Asp Gly Tyr Leu Ile Gln Gly Asp Ala 690 695 700 Thr Gln Glu Ile Leu Val Asp Gly Asn Gln Glu Val Ile Phe Leu Tyr 705 710 715 720 Ser Lys Val Ala Thr Asp Thr Thr Asp Thr Thr Asp Thr Thr Asp Thr 725 730 735 Thr Asp Thr Thr Asp Thr Thr Asp Thr Thr Asp Thr Thr Asp Thr Thr 740 745 750 Asp Thr Thr Asp Thr Thr Asp Thr Thr Glu Thr Ser Glu Ala Thr Glu 755 760 765 Asn Ser Gly Asn Lys Ser Lys Val Ala Val Val Asn Asn Asn Ser Asn 770 775 780 Thr Thr Gly Gly Ser Thr Asn Ser Ser Gly Lys Ala Leu Pro Gln Thr 785 790 795 800 Gly Glu Lys Asn Gln Leu Ala Ile Ser Ile Leu Gly Val Ala Val Ile 805 810 815 Val Ala Val Val Gly Ala Val Leu Tyr Lys Lys Lys Ala 820 825 20475PRTCellulomonas fimi 20Met Ala Asp Glu Thr Ile Ala Ile Val Asp Ala Asp Ala Thr Ala Glu 1 5 10 15 Thr Arg Ser Leu Leu Ser Tyr Leu Asp Gly Val Arg Gly Glu Gly Ile 20 25 30 Leu Phe Gly His Gln His Thr Thr Ser Phe Gly Leu Thr Thr Gly Pro 35 40 45 Thr Asp Gly Thr Thr Ser Asp Val Lys Asn Val Thr Gly Asp Phe Pro 50 55 60 Ala Val Phe Gly Trp Asp Thr Leu Ile Ile Glu Gly Asn Glu Arg Pro 65 70 75 80 Gly Leu Ala Glu Asn Thr Arg Asp Glu Asn Ile Ala Leu Phe Ala Asp 85 90 95 Tyr Ile Arg Lys Ala Asp Ala Ile Gly Gly Val Asn Thr Val Ser Ala 100 105 110 His Val Glu Asn Phe Val Thr Gly Gly Ser Phe Tyr Asp Thr Ser Gly 115 120 125 Asp Thr Leu Arg Ala Val Leu Pro Gly Gly Ser His His Ala Glu Leu 130 135 140 Val Ala Tyr Leu Asp Asp Ile Ala Glu Leu Ala Asp Ala Ser Arg Arg 145 150 155 160 Asp Asp Gly Thr Leu Ile Pro Ile Val Phe Arg Pro Trp His Glu Asn 165 170 175 Ala Gly Ser Trp Phe Trp Trp Gly Ala Ala Tyr Gly Ser Pro Gly Glu 180 185 190 Tyr Gln Glu Leu Tyr Arg Phe Thr Val Glu Tyr Leu Arg Asp Val Lys 195 200 205 Gly Val Ser Asn Phe Leu Tyr Ala Trp Gly Pro Gly Gly Gly Phe Gly 210 215 220 Gly Asn Arg Asp Val Tyr Leu Arg Thr Tyr Pro Gly Asp Ala Phe Val 225 230 235 240 Asp Val Leu Gly Leu Asp Thr Tyr Asp Ser Thr Gly Ser Asp Ala Phe 245 250 255 Leu Ala Gly Leu Val Ala Asp Leu Arg Met Ile Ala Glu Ile Ala Asp 260 265 270 Glu Lys Gly Lys Val Ser Ala Phe Thr Glu Phe Gly Val Ser Gly Gly 275 280 285 Val Gly Thr Asn Gly Ser Ser Pro Ala Gln Trp Phe Thr Lys Val Leu 290 295 300 Ala Ala Ile Lys Ala Asp Pro Val Ala Ser Arg Asn Ala Tyr Met Glu 305 310 315 320 Thr Trp Ala Asn Phe Asp Ala Gly Gln His Phe Val Pro Val Pro Gly 325 330 335 Asp Ala Leu Leu Glu Asp Phe Gln Ala Tyr Ala Ala Asp Pro Phe Thr 340 345 350 Leu Phe Ala Ser Glu Val Thr Gly Ala Phe Asp Arg Thr Val Ala Ala 355 360 365 Ala Pro Ala Gln Pro Val Val His Ile Ala Ser Pro Ala Asp Gly Ala 370 375 380 Arg Val Ala Ser Ala Pro Thr Thr Val Arg Val Arg Val Gly Gly Thr 385 390 395 400 Asp Val Gln Ser Val Thr Val Glu Val Ala Gln Gly Gly Thr Val Val 405 410 415 Asp Thr Leu Asp Leu Ala Tyr Asp Gly Ala Leu Trp Trp Thr Ala Pro 420 425 430 Trp Ser Pro Thr Ser Ala Gln Leu Asp Asn Ser Thr Tyr Thr Val Thr 435 440 445 Ala Thr Ala Thr Thr Ala Ala Gly Thr Leu Asp Val Thr Asn Glu Val 450 455 460 Ala Ala Ala Leu Glu His His His His His His 465 470 475 211008PRTGeobacillus tepidamans 21Lys Lys Gln Lys Asn Pro Ser Lys Pro Asn Ser Lys Arg Val Glu Asn 1 5 10 15 Leu Val Asp Pro Leu Ala Thr Asp Asp Thr Lys Ser Leu Phe Ala Tyr 20 25 30 Leu Lys Asp Val Arg Gly Lys Gln Val Leu Phe Gly His Gln His Ala 35 40 45 Ile Asp Glu Gly Leu Thr Leu Ile Gly Ser Lys Glu Leu Glu Ser Glu 50 55 60 Val Lys Asn Ser Val Gly Asp Phe Pro Ala Val Phe Gly Trp Asp Thr 65 70 75 80 Leu Ser Leu Glu Gly Lys Glu Lys Pro Gly Val Pro Asn Asp Pro Lys 85 90 95 Gln Ser Arg Ala Asn Leu Val Ala Ser Met Lys Lys Val His Lys Leu 100 105 110 Gly Gly Ile Ile Ala Leu Ser Ala His Met Pro Asn Phe Val Thr Gly 115 120 125 Gly Ser Phe Asn Asp Thr Thr Gly Asn Val Val Glu His Ile Leu Pro 130 135 140 Gly Gly Asp Lys Asn Ala Glu Phe Asn Ser Phe Leu Asp Asn Ile Ala 145 150 155 160 Gln Phe Ala Lys Glu Leu Lys Asp Asp Lys Gly Lys Gln Ile Pro Ile 165 170 175 Leu Phe Arg Pro Phe His Glu Gln Asn Gly Ser Trp Phe Trp Trp Gly 180 185 190 Ala Lys Thr Thr Thr Pro Ser Gln Tyr Ile Glu Ile Tyr Arg Tyr Thr 195 200 205 Val Glu Tyr Leu Arg Asp Lys Lys Gly Val His Asn Phe Leu Tyr Val 210 215 220 Tyr Ser Pro Asn Gly Thr Phe Gly Gly Ser Glu Ala Asn Tyr Leu Thr 225 230 235 240 Thr Tyr Pro Gly Asp Asp Tyr Val Asp Ile Leu Gly Met Asp Gln Tyr 245 250 255 Asp Asn Gln Ser Asn Pro Gly Thr Thr Gln Phe Leu Thr Asn Leu Val 260 265 270 Lys Asp Leu Glu Met Ile Ser Lys Leu Ala Asp Thr Lys Gly Lys Ile 275 280 285 Ala Ala Phe Ser Glu Phe Gly Tyr Ser Pro Gln Gly Met Lys Thr Thr 290 295 300 Gly Asn Gly Asp Leu Lys Trp Phe Thr Lys Val Leu Asn Ala Ile Lys 305 310 315 320 Ala Asp Arg Asn Ala Lys Arg Ile Ala Tyr Met Gln Thr Trp Ala Asn 325 330 335 Phe Gly Leu Asn Gly Asn Leu Phe Val Pro Tyr Asn Asp Ala Pro Asn 340 345 350 Gly Leu Gly Asp His Glu Leu Leu Pro Asp Phe Ile Asn Tyr Tyr Lys 355 360 365 Asp Pro Tyr Thr Ala Phe Leu Arg Glu Val Lys Gly Val Tyr Asn Asn 370 375 380 Lys Val Glu Ala Ala Lys Glu Gln Pro Phe Met His Ile Ala Ser Pro 385 390 395 400 Thr Asp Asn Ala Thr Val Lys Thr Ala Thr Thr Lys Ile Arg Val Arg 405 410 415 Val Leu Asn Gln Lys Pro Ser Lys Val Val Tyr Val Val Glu Gly Ser 420 425 430 Ser Lys Glu Val Pro Met Lys Leu Asp Ala Asp Gly Tyr Tyr Ser Ala 435 440 445 Asn Trp Ser Pro Val Ser Lys Phe Asn Gly Lys Ser Val Lys Ile Thr 450 455 460 Val Lys Ser Tyr Met Pro Asn Lys Thr Val Met Lys Gln Thr Val Asn 465 470 475 480 Val Phe Val Lys Val Pro Glu Ile Leu Ile Lys Gln Phe Thr Phe Asp 485 490 495 Arg Asp Ile Lys Gly Ile Arg Asn Ile Gly Thr Trp Pro Asp Thr Ile 500 505 510 Lys Thr Asn Phe Glu His Ala Arg Leu Asn Gly Asn Gly Lys Leu Lys 515 520 525 Ile Asn Ile Thr Gly Met Val Arg Thr Asp Thr Trp Gln Glu Ile Lys 530 535 540 Leu Glu Leu Ser Asn Ile Lys Asp Ile Val Pro Leu Ser Asn Val Asn 545 550 555 560 Arg Val Lys Phe Asp Val Leu Val Pro Val Ser Ala Gly Gln Gln Asn 565 570 575 Ala Asn Ala Ser Leu Arg Gly Ile Ile Met Leu Pro Pro Asp Trp Asn 580 585 590 Glu Lys Tyr Gly Met Thr Thr Thr Glu Lys Ala Leu Ala Asn Leu Gln 595 600 605 Thr Val Thr Ile Asn Arg Val Lys Tyr Ala Glu Phe Pro Val Met Ile 610 615 620 Asp Leu Asn Asp Pro Ala Lys Leu Ser Ala Ala Lys Gly Leu Val Leu 625 630 635 640 Ser Ile Val Gly Asn Gly Leu Glu Leu Asn Gly Ala Val Tyr Val Asp 645 650 655 Asn Ile Lys Leu Phe Ser Thr Tyr Thr Glu Thr Pro Thr Asp Pro Ala 660 665 670 Leu Val Asp Asp Phe Glu Ser Tyr Gln Gly Ser Asn Ala Val Leu Gln 675 680 685 Gln Lys Phe Val Lys Ala Gly Gly Asp Thr Ile Thr Val Ser Leu Asp 690 695 700 Gly Ser His Lys Ser Ser Gly Thr Tyr Ala Met Lys Val Asp Tyr Thr 705 710 715 720 Leu Ala Gly Ser Gly Tyr Ala Gly Val Thr Lys Ser Leu Gly Gly Val 725 730 735 Asp Trp Ser Arg Phe Asn Lys Leu Lys Phe Trp Leu Thr Pro Asp Gly 740 745 750 Lys Asp Gln Lys Leu Val Ile Gln Leu Arg Val Asp Gly Val Tyr Tyr 755 760 765 Glu Ala Tyr Pro Ser Leu Ala Ser Thr Thr Pro Gly Trp Val Glu Leu 770 775 780 His Phe Asn Asp Phe Thr Val Ala Pro Trp Asp Thr Ala Asn Leu Gly 785 790 795 800 Lys Lys Leu Asn Lys Ile Ser Leu Lys Asn Val Gln Asp Phe Ala Ile 805 810 815 Tyr Val Asn Ser Lys Asn Gly Thr Thr Leu

Ser Ser Thr Leu Tyr Phe 820 825 830 Asp Asp Ile Lys Ala Ile Tyr Asp Ala Thr Ala Ala Ser Val Pro Asn 835 840 845 Gly Gly Thr Gly Pro Gly Ser Thr Pro Glu Gln Pro Gly Thr Leu Tyr 850 855 860 Asp Phe Glu Thr Gly Val Gln Gly Trp Glu Val Glu Gln Asn Gln Ala 865 870 875 880 Asn Ala Thr Thr Pro Thr Ile Thr Thr Asp Ala Ala Ala Lys Gly Thr 885 890 895 His Ser Leu Thr Ser Thr Phe Asp Leu Thr Lys Thr Gly Gly Phe Glu 900 905 910 Leu Thr Lys Val Gln Val Val Asp Leu Ser Ala Val Lys Thr Ile Ser 915 920 925 Ala Lys Val Lys Ile Ser Thr Gly Thr Ala Asn Ala Arg Leu Tyr Ile 930 935 940 Lys Thr Gly Ser Asn Trp Gln Trp His Asp Ser Gly Met Val Ala Val 945 950 955 960 Asp Ser Ser Glu Phe Lys Thr Leu Thr Ile Ser Leu Asn Pro Ala Trp 965 970 975 Gly Ile Asp Asn Val Lys Ser Ile Gly Val Lys Ile Glu Pro Thr Ser 980 985 990 Gly Thr Gly Asn Ala Ser Val Tyr Val Asp Asp Val Ala Leu Ser Glu 995 1000 1005 221010PRTThermoanaerobacterium thermosaccharolyticum 22Asn Asn Ser Gln Asn Asn Ser Asn Asn Gly Ser Thr Ile Lys Glu Ile 1 5 10 15 Asn Leu Val Asp Pro Asn Ala Thr Thr Glu Thr Lys Glu Leu Phe Val 20 25 30 Tyr Leu Asn Asp Ile Arg Gly Lys Glu Val Leu Phe Gly His Gln His 35 40 45 Asp Thr Asp Glu Gly Ile Thr Ile Thr Ser Gly Ser Asn Glu Leu Gln 50 55 60 Ser Asp Val Lys Asn Asp Val Gly Asp Phe Pro Ala Val Phe Gly Trp 65 70 75 80 Asp Thr Leu Ser Leu Glu Gly Lys Glu Lys Pro Gly Val Pro Asn Asp 85 90 95 Pro Val Lys Ser Arg Glu Asn Leu Ile Ala Ala Val Lys Lys Ile His 100 105 110 Glu Met Gly Gly Ile Phe Thr Leu Ser Ala His Met Pro Asn Phe Val 115 120 125 Thr Gly Gly Ser Phe Asn Asp Val Ser Asp Asp Val Val Asp Lys Ile 130 135 140 Leu Pro Gly Gly Glu Tyr Asn Ser Lys Phe Asn Glu Phe Leu Asp Asn 145 150 155 160 Ile Ala Leu Phe Ala Asn Asn Leu Lys Asp Asp Asn Gly Asn Leu Ile 165 170 175 Pro Ile Leu Phe Arg Pro Phe His Glu Gln Asn Gly Gly Trp Phe Trp 180 185 190 Trp Gly Ala Lys Thr Thr Thr Pro Ser Gln Tyr Ile Glu Leu Tyr Arg 195 200 205 Tyr Thr Val Glu Tyr Leu Arg Asp Lys Lys Gly Val His Asn Ile Leu 210 215 220 Tyr Val Tyr Ser Pro Asn Gly Pro Phe Asn Gly Asn Glu Glu Asn Tyr 225 230 235 240 Leu Val Thr Tyr Pro Gly Asp Ile Tyr Val Asp Val Leu Gly Met Asp 245 250 255 Gln Tyr Asp Asn Ile Asp Asn Pro Gly Thr Lys Gln Phe Leu Ser Ser 260 265 270 Leu Val Asn Asp Leu Ser Met Ile Ser Lys Leu Ala Asp Ser Lys Gly 275 280 285 Lys Ile Ala Thr Leu Ser Glu Phe Gly Tyr Ser Pro Gln Gly Met Lys 290 295 300 Val Thr Gly Asn Gly Asp Leu Ser Trp Phe Thr Asp Val Leu Asn Ala 305 310 315 320 Ile Lys Ser Asn Ser Asn Ala Arg Arg Ile Ala Tyr Met Leu Thr Trp 325 330 335 Ala Asn Phe Gly Leu Asn Gly Asn Leu Phe Val Pro Tyr Lys Asn Ala 340 345 350 Pro Asn Leu Gly Asp His Glu Leu Leu Pro Asp Phe Ile Lys Phe Tyr 355 360 365 Gln Asp Pro Tyr Thr Ala Phe Leu Asn Asp Ile Lys Gly Ala Asn Leu 370 375 380 Thr Thr Asp Val Val Val Asn Pro Gly Lys Ser Phe Met His Ile Val 385 390 395 400 Thr Pro Thr Asp Asn Ser Glu Ile Thr Thr Asn Thr Thr Lys Ile Arg 405 410 415 Val Arg Ile Leu Asn Asp Thr Pro Thr Lys Val Val Tyr Lys Val Asn 420 425 430 Asp Ser Asn Glu Glu Ile Pro Met Thr Leu Asp Gln Asp Gly Tyr Tyr 435 440 445 Ser Gln Asp Trp Ser Pro Ser Tyr Gln Asp Asn Gly Lys Thr Ala Lys 450 455 460 Ile Thr Val Ile Ala Tyr Asn Gly Asp Ser Ile Glu Phe Glu Gln Ser 465 470 475 480 Val Asn Val Phe Val Lys Val Pro Glu Ile Leu Val Lys Asp Tyr Thr 485 490 495 Phe Asp Thr Gly Ile Asp Gly Ile Gln Asn Asn Gly Thr Tyr Pro Glu 500 505 510 Ser Met Ser Leu Asn Ile Gly His Ala Val Leu Ala Gly Asp Gly Lys 515 520 525 Leu Glu Met Thr Val Thr Gly Met Thr Tyr Ala Asp Ser Trp Gln Glu 530 535 540 Leu Lys Leu Gln Leu Thr Asn Ile Asp Asp Val Leu Pro Tyr Val Asn 545 550 555 560 Arg Val Lys Phe Asp Val Leu Ile Pro Ala Thr Ala Ala Ser Ala Asn 565 570 575 Pro Asp Ala Thr Val Arg Gly Ile Ala Met Leu Pro Asp Asp Trp Asp 580 585 590 Thr Lys Tyr Gly Met Thr Thr Thr Glu Lys Lys Ile Thr Asp Leu Ser 595 600 605 Thr Glu Ser Ile Asp Gly Ile Gln Tyr Ala Tyr Phe Pro Val Thr Ile 610 615 620 Asp Leu Asp Ser Ser Lys Val Ser Ser Ala Lys Gly Leu Ala Ile Ser 625 630 635 640 Val Val Gly Asn Gly Leu Asn Phe Asp Gly Thr Gly Glu Ile Tyr Val 645 650 655 Asp Asn Ile Gln Leu Ile Asn Ala Phe Val Glu Thr Pro Thr Asp Pro 660 665 670 Ser Leu Val Asp Asp Phe Glu Ser Tyr Gln Gly Asn Asp Ala Ala Leu 675 680 685 Gln Ser Lys Trp Ile Lys Ala Ser Gly Asp Asp Ile Ser Val Ser Leu 690 695 700 Thr Asn Asp Asn Ala Ala Asp Gly Met Tyr Ala Met Lys Val Asp Tyr 705 710 715 720 Lys Leu Gly Ser Ser Gly Tyr Ala Gly Val Thr Lys Thr Leu Gly Gly 725 730 735 Val Asp Trp Ser Gly Tyr Asn Lys Leu Lys Phe Tyr Leu Val Pro Asp 740 745 750 Gly Ser Asn Gln Lys Leu Val Ile Gln Ile Lys Val Asn Gly Ile Tyr 755 760 765 Tyr Glu Ala Tyr Pro Ser Leu Ser Asp Ser Thr Pro Arg Trp Glu Glu 770 775 780 Ile Gly Phe Asn Ser Phe Thr Val Ala Pro Trp Asp Thr Gln Asp Gln 785 790 795 800 Gly Lys Val Ile Thr Lys Glu Asp Leu Lys Asn Val Gln Glu Leu Ser 805 810 815 Ile Tyr Ile Asn Asp Ala Gly Gly Ser Lys Ser Gly Thr Leu Tyr Phe 820 825 830 Asp Gly Ile Arg Ala Ile Asn Asp Gly Thr Gly Gly Val Pro Asn Gly 835 840 845 Gly Ser Gly Ser Asn Ser Thr Pro Ala Gln Pro Gly Val Leu Tyr Asp 850 855 860 Phe Glu Asn Gly Thr Asp Gly Trp Thr Val Asp Gln Asn Asn Ala Asn 865 870 875 880 Ala Thr Ala Thr Ser Ile Thr Thr Asp Phe Ala Ser Ser Gly Thr His 885 890 895 Ser Leu Thr Ser Asn Phe Asp Leu Ser Lys Thr Asp Gly Phe Glu Ile 900 905 910 Asp Lys Val Gln Ala Ile Asp Leu Ser Ala Val Lys Lys Ile Ser Ile 915 920 925 Asp Val Lys Leu Ser Asn Gly Thr Ala Thr Ala Thr Leu Tyr Ile Lys 930 935 940 Thr Gly Ser Ser Trp Thr Trp Tyr Asp Ser Gly Trp Gln Pro Ile Asn 945 950 955 960 Ser Gly Gly Phe Thr Thr Leu Ser Ile Asp Leu Asp Pro Ser Lys Ile 965 970 975 Asn Asn Leu Glu Asn Val Gln Ser Ile Gly Val Lys Ile Val Pro Asp 980 985 990 Ser Gly Gln Thr Gly Asn Ser Asn Val Tyr Leu Asp Asn Val Val Leu 995 1000 1005 Ser Asn 1010 23632PRTStreptomyces sviceus 23Gly Thr Pro Thr Pro Val Arg Ile Val Asp Asp Glu Ala Thr Pro Ala 1 5 10 15 Thr Arg Ala Leu Phe Ala Tyr Leu Lys Arg Gln Gln Gly Lys Gly Ile 20 25 30 Leu Phe Gly His Gln His Asp Leu Thr Tyr Gly Phe Thr Phe Thr Thr 35 40 45 Pro Asn Gly Arg Ala Ser Asp Thr Arg Ala Gly Val Gly Asp Tyr Pro 50 55 60 Ala Val Phe Gly Trp Asp Thr Leu Ile Leu Asp Gly Asp Glu Arg Pro 65 70 75 80 Gly Ala Glu Gly Ala Ser Glu Ala Glu Asn Ile Ala Ala Leu Ser Arg 85 90 95 Cys Ile Arg Gln Gly Asp Ala Arg Gly Gly Ile Asn Thr Leu Ser Ala 100 105 110 His Met Pro Asn Phe Val Thr Gly Lys Asn Phe His Asp Thr Thr Gly 115 120 125 Arg Val Val Ser Gln Ile Leu Pro Gly Gly Ala Lys His Ala His Phe 130 135 140 Asn Ala Phe Leu Asp Arg Ile Ala Lys Ala Val Lys Arg Ala Leu Arg 145 150 155 160 Pro Asp Gly Thr Ala Ile Pro Val Val Phe Arg Pro Phe His Glu Asn 165 170 175 Asn Gly Ala Trp Phe Trp Trp Gly Ala Gly His Thr Thr Pro Ala Glu 180 185 190 Phe Ile Glu Leu Phe Arg Tyr Thr Val Glu Tyr Leu Arg Asp Thr Arg 195 200 205 Gly Val His Asn Leu Leu Tyr Ala Tyr Ser Pro Asn Ser Ser Phe Ala 210 215 220 Gly Asp Pro Ala Asp Tyr Leu Lys Thr Tyr Pro Gly Asp Arg Phe Val 225 230 235 240 Asp Val Leu Gly Phe Asp Ser Tyr Asp Glu Asn Ala Gly Pro Thr Pro 245 250 255 Trp Leu Asp Ala Val Val Lys Asp Leu Ala Met Val Val Arg Leu Ala 260 265 270 Asn Glu Arg Gly Lys Ala Pro Ala Phe Thr Glu Phe Gly Glu Gly Gly 275 280 285 Thr Glu Val Arg Asn Gln Gln Trp Phe Thr Gln Leu Ala Gln Ala Ile 290 295 300 Glu Ala Asp Pro Leu Ala Arg Gln Val Thr Tyr Met Leu Thr Trp Ala 305 310 315 320 Asn Phe Gly Gly Thr Lys Arg Ala Tyr Val Pro Tyr Pro Gly His Leu 325 330 335 Leu Phe Pro Asp Phe Val Lys Tyr Glu Gln Asp Pro Tyr Thr Leu Phe 340 345 350 Ala Ala Asp Leu Arg Gly Val Tyr Ser Ala His Thr Thr Ala Val Lys 355 360 365 Asn Ala Pro Phe Leu His Leu Val Thr Pro Thr Asp Arg Gln Arg Val 370 375 380 Ala Ala Pro Arg Thr Thr Ile Arg Val Arg Val Thr Pro Ala Arg Ala 385 390 395 400 Ser Gln Val Thr Tyr Ser Val Asn Gly Gly Arg Ala Arg Lys Leu Cys 405 410 415 Leu Asp Thr Asp Gly Phe Tyr Ser Gly Asp Trp Thr Ile Asp Pro Ala 420 425 430 Leu Arg Asn Asn Arg Ser Val Thr Leu Thr Val Ser Thr Arg Leu Asp 435 440 445 Gly Lys Thr Leu Thr Asp Ser Ala Val Val Leu Leu Gly Glu Leu Thr 450 455 460 Pro Leu Pro Val Gly Trp Val Asp Asp Phe Glu Gly Tyr Ala Ala Asp 465 470 475 480 Asn Thr Ala Leu Ser Gln Ala Tyr Thr His Val Asn Ser His Thr Leu 485 490 495 Thr Leu Ser Ala Asp His Lys Ser Ser Gly Ser Tyr Gly Leu Ala Tyr 500 505 510 Ala Tyr Asp Phe Thr Gly Ser Glu Tyr Thr Gly Ala Gly Lys Pro Leu 515 520 525 Asp Ala Asp Trp Ser Ala Phe Thr Ser Leu Ala Leu Trp Leu Arg Gly 530 535 540 Asp Gly Ser Ala Asn Gly Gly Ala Leu Gln Ile Val Ala Asp Gly Val 545 550 555 560 Asp Phe Trp Tyr Gln Ile Pro Leu Ser Asp Thr Ser Gly Gln Asp Val 565 570 575 Arg Ala Pro Phe Ser Glu Phe Thr Pro Ala Pro Trp Asp Thr Glu His 580 585 590 Thr Gly Ala Val Leu Asp Ala Ala His Leu Ala Glu Val Thr Ala Phe 595 600 605 Asn Leu Tyr Leu Val His Gly Ser Gly Ala Ala Thr Lys Gly Thr Val 610 615 620 Tyr Val Asp Asn Ile Arg Ala Glu 625 630 24580PRTStreptomyces scabiei 24Gly Thr Pro Thr Pro Val Arg Ile Val Asp Asp Arg Ala Thr Pro Ala 1 5 10 15 Thr Arg Ala Leu Phe Ala Tyr Leu Arg Arg Gln Arg Gly Arg Gly Ile 20 25 30 Leu Phe Gly His Gln His Asp Leu Thr Tyr Gly Phe Thr Phe Thr Thr 35 40 45 Pro Asp Gly Arg Ala Ser Asp Thr Arg Ala Ala Val Gly Asp Tyr Pro 50 55 60 Ala Val Phe Gly Trp Asp Thr Leu Val Leu Asp Gly Asp Glu Arg Pro 65 70 75 80 Gly Thr Glu Asp Ala Thr Asp Ala Glu Asn Ile Ala Ala Leu Ser Arg 85 90 95 Cys Ile Arg Gln Gly Asp Ala Arg Gly Gly Ile Asn Thr Leu Ser Ala 100 105 110 His Met Pro Asn Phe Val Thr Gly Lys Asp Phe Tyr Asp Thr Arg Gly 115 120 125 Arg Val Val Gly Gln Ile Leu Pro Gly Gly Ala Lys His Ala Arg Phe 130 135 140 Asn Arg Phe Leu Asp Arg Val Ala Lys Ala Val Lys Gly Ala Arg Arg 145 150 155 160 Pro Asp Gly Thr Leu Ile Pro Val Ile Phe Arg Pro Phe His Glu Asn 165 170 175 Asn Gly Gly Trp Phe Trp Trp Gly Ala Gly His Thr Thr Ser Gly Glu 180 185 190 Phe Ile Glu Leu Phe Arg Tyr Thr Val Glu Tyr Leu Arg Asp Val Lys 195 200 205 Gly Val His Asn Leu Leu Tyr Ala Tyr Ser Pro Asn Ala Ser Leu Gly 210 215 220 Gly Asp Pro Ala Ala Tyr Leu Arg Thr Tyr Pro Gly Asp Arg Phe Val 225 230 235 240 Asp Val Leu Gly Tyr Asp Ser Tyr Asp Glu Gly Ala Gly Pro Thr Pro 245 250 255 Trp Leu Asp Gly Leu Val Arg Asp Leu Ala Met Val Val Arg Leu Ala 260 265 270 Asn Glu Arg Ala Tyr Val Pro Tyr Pro Gly His Ala Leu Leu Pro Asp 275 280 285 Phe Val Arg Phe His Gln Asp Pro Phe Thr Leu Phe Ala Ala Asp Val 290 295 300 Arg Gly Val Phe Ala Ala Arg Thr Thr Ala Val Arg Asn Gly Pro Ser 305 310 315 320 Leu His Leu Val Thr Pro Thr Asp Arg Gln Arg Val Thr Ala Ala Arg 325 330 335 Thr Thr Val Arg Val Arg Val Thr Pro Ala Arg Ala Ser Arg Val Thr 340 345 350 Tyr Ser Val Asp Gly Gly Pro Ala Arg Arg Leu Arg Leu Asp Ala Asp 355 360 365 Gly Tyr His Ser Gly Val Trp Ser Ile Gly Pro Ala Leu Arg Arg Lys 370 375 380 Gly Ser Ala Thr Leu Thr Val Arg Ala Arg Ala Gly Gly Glu Thr Leu 385 390 395 400 Thr Asp Ser Ala Val Val Leu Leu Gly Glu Ala Ala Pro Leu Pro Ala 405 410 415 Gly Trp Ile Asp Asp Phe Glu Gly Tyr Ala Gly Asp Asp Val Ala Leu 420 425 430 Gly Glu Ala Tyr Thr His Leu Asn Thr His Thr Leu Gly Leu Ser Arg 435 440 445 Glu His Lys Ser Ser Gly Ser Tyr Gly Leu Ala Tyr Ala Tyr Asp Phe 450 455

460 Thr Ala Ala Glu Phe Thr Gly Ile Gly Arg Pro Val Val Ala Asp Trp 465 470 475 480 Ser Ala Phe Thr Ser Leu Ala Leu Trp Leu Arg Gly Asp Gly Ser Glu 485 490 495 Asn Ala Gly Ala Leu Glu Ile Val Ala Asp Gly Ile Pro Phe Gln Tyr 500 505 510 Arg Phe Ala Leu Asp Asp Thr Ser Gly Arg Glu Leu Arg Ala Pro Phe 515 520 525 Gly Glu Phe Gly Pro Ala Pro Trp Asp Thr Gly Asn Ala Gly Ala Val 530 535 540 Leu Asp Ala Ala Arg Leu Ala Lys Val Thr Gly Phe Asn Leu Tyr Leu 545 550 555 560 Gly Arg Ala Ser Glu Thr Val Thr Lys Gly Val Val Tyr Val Asp Ala 565 570 575 Val Arg Ala Glu 580 2538DNAArtificial Sequencesynthetic primer 25actagccgac tagttcacaa gaagggcgtc aacttaac 382641DNAArtificial Sequencesynthetic primer 26cttacgggct cgagttaacc taattcattg tggatatcac g 412746DNAArtificial Sequencesynthetic primer 27cttacgggct cgagttattg gactttttct ccactagaat aataag 462841DNAArtificial Sequencesynthetic primer 28cttacgggct cgagttaccc aaaataacct tcaaaatcat c 412944DNAArtificial Sequencesynthetic primer 29cttacgggct cgagttaaat aggagcgacc tctttttcct cttc 44301053DNABacillus sp. 30tcacaagaag ggcgtcaact taacatggca gatgaggatg cttcaaagta tacgaaggag 60ttatttgctt ttcttcaaga tgtaagtggt tcacaagtgt tatttggaca acagcatgca 120acagatgaag gattaacttt aacaaatcca gctccaagaa caggttccac tcaatctgaa 180gttttcaatg cagttgggga ttatccagct gtgtttggat gggacacgaa tagcctagat 240ggtcgtgaaa agcctggcat tgcaggtaat gtagaacaaa gtataaaaaa tacggctcag 300tccatgaaag tggctcatga tttaggaggg attattacac taagcatgca cccagataat 360tttgtaacag ggggtcctta tggtgataca acagggaatg ttgtaaaaga aattcttcca 420ggtggatcaa aacatgcaga gtttaacgcg tggttggaca atattgctgc gcttgctcac 480gagctgaaag atgagaatgg tgaacctatt ccgatgattt ttcggccatt ccatgaacaa 540acaggatctt ggttttggtg gggagcaagc acaacttcac ccgaacaata taaagcgatt 600tttcgttata cagtagaata tttgcgagat gttaaaggcg taaataatat tttatatggc 660ttttcacctg gggcgggacc tgctggagat gtaaatcgct atttagaaac atatccaggg 720gatgattacg ttgatatttt cggtattgac aattatgaca ataaagacaa tgcagggtca 780gaagcttggt taagtggtat ggtcaaagac ttggcgatga ttagccgatt agctgaacaa 840aaagaaaaag tagcggcttt tactgagtat gggtacagtg caaccggaat taatcgtcaa 900gggaatacat tagactggta cacacgtgta ttagatgcga ttgctgctga tgaagacgca 960cgtaaaatat catacatgtt gacatgggcg aactttggtt ggccgaataa tatgtatgtt 1020ccttatcgtg atatccacaa tgaattaggt taa 105331381PRTArtificial Sequencesynthetic Bsp Man4 sequence 31Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Thr Ser Ser 20 25 30 Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys Tyr 35 40 45 Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln Val 50 55 60 Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr Asn 65 70 75 80 Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala Val 85 90 95 Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp Gly 100 105 110 Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys Asn 115 120 125 Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile Thr 130 135 140 Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly Asp 145 150 155 160 Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys His 165 170 175 Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His Glu 180 185 190 Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro Phe 195 200 205 His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr Ser 210 215 220 Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu Arg 225 230 235 240 Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly Ala 245 250 255 Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly Asp 260 265 270 Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp Asn 275 280 285 Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala Met 290 295 300 Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr Glu 305 310 315 320 Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu Asp 325 330 335 Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala Arg 340 345 350 Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn Asn 355 360 365 Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly 370 375 380 32350PRTBacillus sp. 32Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys 1 5 10 15 Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln 20 25 30 Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr 35 40 45 Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala 50 55 60 Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp 65 70 75 80 Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys 85 90 95 Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile 100 105 110 Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly 115 120 125 Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys 130 135 140 His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His 145 150 155 160 Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro 165 170 175 Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr 180 185 190 Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu 195 200 205 Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly 210 215 220 Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly 225 230 235 240 Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp 245 250 255 Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala 260 265 270 Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr 275 280 285 Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu 290 295 300 Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala 305 310 315 320 Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn 325 330 335 Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly 340 345 350 331428DNABacillus sp. 33tcacaagaag ggcgtcaact taacatggca gatgaggatg cttcaaagta tacgaaggag 60ttatttgctt ttcttcaaga tgtaagtggt tcacaagtgt tatttggaca acagcatgca 120acagatgaag gattaacttt aacaaatcca gctccaagaa caggttccac tcaatctgaa 180gttttcaatg cagttgggga ttatccagct gtgtttggat gggacacgaa tagcctagat 240ggtcgtgaaa agcctggcat tgcaggtaat gtagaacaaa gtataaaaaa tacggctcag 300tccatgaaag tggctcatga tttaggaggg attattacac taagcatgca cccagataat 360tttgtaacag ggggtcctta tggtgataca acagggaatg ttgtaaaaga aattcttcca 420ggtggatcaa aacatgcaga gtttaacgcg tggttggaca atattgctgc gcttgctcac 480gagctgaaag atgagaatgg tgaacctatt ccgatgattt ttcggccatt ccatgaacaa 540acaggatctt ggttttggtg gggagcaagc acaacttcac ccgaacaata taaagcgatt 600tttcgttata cagtagaata tttgcgagat gttaaaggcg taaataatat tttatatggc 660ttttcacctg gggcgggacc tgctggagat gtaaatcgct atttagaaac atatccaggg 720gatgattacg ttgatatttt cggtattgac aattatgaca ataaagacaa tgcagggtca 780gaagcttggt taagtggtat ggtcaaagac ttggcgatga ttagccgatt agctgaacaa 840aaagaaaaag tagcggcttt tactgagtat gggtacagtg caaccggaat taatcgtcaa 900gggaatacat tagactggta cacacgtgta ttagatgcga ttgctgctga tgaagacgca 960cgtaaaatat catacatgtt gacatgggcg aactttggtt ggccgaataa tatgtatgtt 1020ccttatcgtg atatccacaa tgaattaggt ggagaccatg agttattacc ggactttgaa 1080gctttccatg cggatgacta cacagcattt cgagatgaga taaaaggaaa gatatataat 1140actggaaagg aatataccgt ttctcctcat gagccgttta tgtatgttat atctccgatt 1200acaggttcta cagtgacaag cgaaacggta acaatccaag caaaagtagc gaatgacgaa 1260cacgcaagag tcactttcag ggtcgatggt tctagtttgg aagaagaaat ggttttcaat 1320gatgacactt tatattatac aggttctttt acaccagatg cagcagtgaa tggcggagct 1380gttgatgtga ttgtagctta ttattctagt ggagaaaaag tccaataa 142834506PRTArtificial Sequencesynthetic Bsp Man4 sequence 34Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Thr Ser Ser 20 25 30 Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys Tyr 35 40 45 Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln Val 50 55 60 Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr Asn 65 70 75 80 Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala Val 85 90 95 Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp Gly 100 105 110 Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys Asn 115 120 125 Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile Thr 130 135 140 Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly Asp 145 150 155 160 Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys His 165 170 175 Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His Glu 180 185 190 Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro Phe 195 200 205 His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr Ser 210 215 220 Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu Arg 225 230 235 240 Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly Ala 245 250 255 Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly Asp 260 265 270 Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp Asn 275 280 285 Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala Met 290 295 300 Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr Glu 305 310 315 320 Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu Asp 325 330 335 Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala Arg 340 345 350 Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn Asn 355 360 365 Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly Gly Asp His 370 375 380 Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp Tyr Thr Ala 385 390 395 400 Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly Lys Glu Tyr 405 410 415 Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser Pro Ile Thr 420 425 430 Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala Lys Val Ala 435 440 445 Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp Gly Ser Ser Leu 450 455 460 Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr Thr Gly Ser 465 470 475 480 Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp Val Ile Val 485 490 495 Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln 500 505 35475PRTBacillus sp. 35Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys 1 5 10 15 Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln 20 25 30 Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr 35 40 45 Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala 50 55 60 Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp 65 70 75 80 Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys 85 90 95 Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile 100 105 110 Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly 115 120 125 Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys 130 135 140 His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His 145 150 155 160 Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro 165 170 175 Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr 180 185 190 Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu 195 200 205 Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly 210 215 220 Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly 225 230 235 240 Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp 245 250 255 Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala 260 265 270 Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr 275 280 285 Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu 290 295 300 Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala 305 310 315 320 Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn 325 330 335 Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly Gly Asp 340 345 350 His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp Tyr Thr 355 360 365 Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly Lys Glu 370 375 380 Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser Pro Ile 385 390 395 400 Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala Lys Val 405 410 415 Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp Gly Ser Ser 420 425 430 Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr Thr Gly 435 440 445 Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp Val Ile 450 455

460 Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln 465 470 475 362028DNABacilllus sp. 36tcacaagaag ggcgtcaact taacatggca gatgaggatg cttcaaagta tacgaaggag 60ttatttgctt ttcttcaaga tgtaagtggt tcacaagtgt tatttggaca acagcatgca 120acagatgaag gattaacttt aacaaatcca gctccaagaa caggttccac tcaatctgaa 180gttttcaatg cagttgggga ttatccagct gtgtttggat gggacacgaa tagcctagat 240ggtcgtgaaa agcctggcat tgcaggtaat gtagaacaaa gtataaaaaa tacggctcag 300tccatgaaag tggctcatga tttaggaggg attattacac taagcatgca cccagataat 360tttgtaacag ggggtcctta tggtgataca acagggaatg ttgtaaaaga aattcttcca 420ggtggatcaa aacatgcaga gtttaacgcg tggttggaca atattgctgc gcttgctcac 480gagctgaaag atgagaatgg tgaacctatt ccgatgattt ttcggccatt ccatgaacaa 540acaggatctt ggttttggtg gggagcaagc acaacttcac ccgaacaata taaagcgatt 600tttcgttata cagtagaata tttgcgagat gttaaaggcg taaataatat tttatatggc 660ttttcacctg gggcgggacc tgctggagat gtaaatcgct atttagaaac atatccaggg 720gatgattacg ttgatatttt cggtattgac aattatgaca ataaagacaa tgcagggtca 780gaagcttggt taagtggtat ggtcaaagac ttggcgatga ttagccgatt agctgaacaa 840aaagaaaaag tagcggcttt tactgagtat gggtacagtg caaccggaat taatcgtcaa 900gggaatacat tagactggta cacacgtgta ttagatgcga ttgctgctga tgaagacgca 960cgtaaaatat catacatgtt gacatgggcg aactttggtt ggccgaataa tatgtatgtt 1020ccttatcgtg atatccacaa tgaattaggt ggagaccatg agttattacc ggactttgaa 1080gctttccatg cggatgacta cacagcattt cgagatgaga taaaaggaaa gatatataat 1140actggaaagg aatataccgt ttctcctcat gagccgttta tgtatgttat atctccgatt 1200acaggttcta cagtgacaag cgaaacggta acaatccaag caaaagtagc gaatgacgaa 1260cacgcaagag tcactttcag ggtcgatggt tctagtttgg aagaagaaat ggttttcaat 1320gatgacactt tatattatac aggttctttt acaccagatg cagcagtgaa tggcggagct 1380gttgatgtga ttgtagctta ttattctagt ggagaaaaag tccaagaaga aacaattcgt 1440ttatttgtaa aaattcctga aatgtctttg ttaacattaa cgtttgatga tgatataaac 1500ggaatcaaaa gcaatggaac atggcctgaa gatggtgtaa catctgaaat tgaccacgct 1560attgtagatg gagacggcaa gttgatgttc tctgttcaag gaatgtcacc tactgaaaca 1620tggcaagagc tcaagttaga attaacagaa ctatcagatg tgaacattga tgcggttaag 1680aaaatgaagt ttgacgcgct tatcccagca ggtagtgaag aaggttcagt ccaaggaatc 1740gtacaacttc caccggattg ggagacgaaa tatgggatga atgaaacaac gaagtcaata 1800aaagacttag agactgttac tgttaatgga agcgattata aacggttgga agtgactgtt 1860tctatcgaca atcaaggagg agctacagga atcgctttat cattagtagg atcccaactc 1920gatttgttag aacctgtcta catcgataat attgaacttc taaattcctt tgaagcacca 1980ccagcagatt cttttcttgt tgatgatttt gaaggttatt ttgggtaa 202837706PRTArtificial Sequencesynthetic Bsp Man4 sequence 37Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Thr Ser Ser 20 25 30 Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys Tyr 35 40 45 Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln Val 50 55 60 Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr Asn 65 70 75 80 Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala Val 85 90 95 Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp Gly 100 105 110 Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys Asn 115 120 125 Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile Thr 130 135 140 Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly Asp 145 150 155 160 Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys His 165 170 175 Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His Glu 180 185 190 Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro Phe 195 200 205 His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr Ser 210 215 220 Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu Arg 225 230 235 240 Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly Ala 245 250 255 Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly Asp 260 265 270 Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp Asn 275 280 285 Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala Met 290 295 300 Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr Glu 305 310 315 320 Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu Asp 325 330 335 Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala Arg 340 345 350 Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn Asn 355 360 365 Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly Gly Asp His 370 375 380 Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp Tyr Thr Ala 385 390 395 400 Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly Lys Glu Tyr 405 410 415 Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser Pro Ile Thr 420 425 430 Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala Lys Val Ala 435 440 445 Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp Gly Ser Ser Leu 450 455 460 Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr Thr Gly Ser 465 470 475 480 Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp Val Ile Val 485 490 495 Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu Thr Ile Arg Leu 500 505 510 Phe Val Lys Ile Pro Glu Met Ser Leu Leu Thr Leu Thr Phe Asp Asp 515 520 525 Asp Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro Glu Asp Gly Val 530 535 540 Thr Ser Glu Ile Asp His Ala Ile Val Asp Gly Asp Gly Lys Leu Met 545 550 555 560 Phe Ser Val Gln Gly Met Ser Pro Thr Glu Thr Trp Gln Glu Leu Lys 565 570 575 Leu Glu Leu Thr Glu Leu Ser Asp Val Asn Ile Asp Ala Val Lys Lys 580 585 590 Met Lys Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu Glu Gly Ser Val 595 600 605 Gln Gly Ile Val Gln Leu Pro Pro Asp Trp Glu Thr Lys Tyr Gly Met 610 615 620 Asn Glu Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr Val Thr Val Asn 625 630 635 640 Gly Ser Asp Tyr Lys Arg Leu Glu Val Thr Val Ser Ile Asp Asn Gln 645 650 655 Gly Gly Ala Thr Gly Ile Ala Leu Ser Leu Val Gly Ser Gln Leu Asp 660 665 670 Leu Leu Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu Leu Asn Ser Phe 675 680 685 Glu Ala Pro Pro Ala Asp Ser Phe Leu Val Asp Asp Phe Glu Gly Tyr 690 695 700 Phe Gly 705 38675PRTBacillus sp. 38Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys 1 5 10 15 Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln 20 25 30 Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr 35 40 45 Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala 50 55 60 Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp 65 70 75 80 Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys 85 90 95 Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile 100 105 110 Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly 115 120 125 Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys 130 135 140 His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His 145 150 155 160 Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro 165 170 175 Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr 180 185 190 Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu 195 200 205 Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly 210 215 220 Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly 225 230 235 240 Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp 245 250 255 Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala 260 265 270 Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr 275 280 285 Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu 290 295 300 Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala 305 310 315 320 Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn 325 330 335 Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly Gly Asp 340 345 350 His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp Tyr Thr 355 360 365 Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly Lys Glu 370 375 380 Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser Pro Ile 385 390 395 400 Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala Lys Val 405 410 415 Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp Gly Ser Ser 420 425 430 Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr Thr Gly 435 440 445 Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp Val Ile 450 455 460 Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu Thr Ile Arg 465 470 475 480 Leu Phe Val Lys Ile Pro Glu Met Ser Leu Leu Thr Leu Thr Phe Asp 485 490 495 Asp Asp Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro Glu Asp Gly 500 505 510 Val Thr Ser Glu Ile Asp His Ala Ile Val Asp Gly Asp Gly Lys Leu 515 520 525 Met Phe Ser Val Gln Gly Met Ser Pro Thr Glu Thr Trp Gln Glu Leu 530 535 540 Lys Leu Glu Leu Thr Glu Leu Ser Asp Val Asn Ile Asp Ala Val Lys 545 550 555 560 Lys Met Lys Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu Glu Gly Ser 565 570 575 Val Gln Gly Ile Val Gln Leu Pro Pro Asp Trp Glu Thr Lys Tyr Gly 580 585 590 Met Asn Glu Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr Val Thr Val 595 600 605 Asn Gly Ser Asp Tyr Lys Arg Leu Glu Val Thr Val Ser Ile Asp Asn 610 615 620 Gln Gly Gly Ala Thr Gly Ile Ala Leu Ser Leu Val Gly Ser Gln Leu 625 630 635 640 Asp Leu Leu Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu Leu Asn Ser 645 650 655 Phe Glu Ala Pro Pro Ala Asp Ser Phe Leu Val Asp Asp Phe Glu Gly 660 665 670 Tyr Phe Gly 675 392553DNABacillus sp. 39tcacaagaag ggcgtcaact taacatggca gatgaggatg cttcaaagta tacgaaggag 60ttatttgctt ttcttcaaga tgtaagtggt tcacaagtgt tatttggaca acagcatgca 120acagatgaag gattaacttt aacaaatcca gctccaagaa caggttccac tcaatctgaa 180gttttcaatg cagttgggga ttatccagct gtgtttggat gggacacgaa tagcctagat 240ggtcgtgaaa agcctggcat tgcaggtaat gtagaacaaa gtataaaaaa tacggctcag 300tccatgaaag tggctcatga tttaggaggg attattacac taagcatgca cccagataat 360tttgtaacag ggggtcctta tggtgataca acagggaatg ttgtaaaaga aattcttcca 420ggtggatcaa aacatgcaga gtttaacgcg tggttggaca atattgctgc gcttgctcac 480gagctgaaag atgagaatgg tgaacctatt ccgatgattt ttcggccatt ccatgaacaa 540acaggatctt ggttttggtg gggagcaagc acaacttcac ccgaacaata taaagcgatt 600tttcgttata cagtagaata tttgcgagat gttaaaggcg taaataatat tttatatggc 660ttttcacctg gggcgggacc tgctggagat gtaaatcgct atttagaaac atatccaggg 720gatgattacg ttgatatttt cggtattgac aattatgaca ataaagacaa tgcagggtca 780gaagcttggt taagtggtat ggtcaaagac ttggcgatga ttagccgatt agctgaacaa 840aaagaaaaag tagcggcttt tactgagtat gggtacagtg caaccggaat taatcgtcaa 900gggaatacat tagactggta cacacgtgta ttagatgcga ttgctgctga tgaagacgca 960cgtaaaatat catacatgtt gacatgggcg aactttggtt ggccgaataa tatgtatgtt 1020ccttatcgtg atatccacaa tgaattaggt ggagaccatg agttattacc ggactttgaa 1080gctttccatg cggatgacta cacagcattt cgagatgaga taaaaggaaa gatatataat 1140actggaaagg aatataccgt ttctcctcat gagccgttta tgtatgttat atctccgatt 1200acaggttcta cagtgacaag cgaaacggta acaatccaag caaaagtagc gaatgacgaa 1260cacgcaagag tcactttcag ggtcgatggt tctagtttgg aagaagaaat ggttttcaat 1320gatgacactt tatattatac aggttctttt acaccagatg cagcagtgaa tggcggagct 1380gttgatgtga ttgtagctta ttattctagt ggagaaaaag tccaagaaga aacaattcgt 1440ttatttgtaa aaattcctga aatgtctttg ttaacattaa cgtttgatga tgatataaac 1500ggaatcaaaa gcaatggaac atggcctgaa gatggtgtaa catctgaaat tgaccacgct 1560attgtagatg gagacggcaa gttgatgttc tctgttcaag gaatgtcacc tactgaaaca 1620tggcaagagc tcaagttaga attaacagaa ctatcagatg tgaacattga tgcggttaag 1680aaaatgaagt ttgacgcgct tatcccagca ggtagtgaag aaggttcagt ccaaggaatc 1740gtacaacttc caccggattg ggagacgaaa tatgggatga atgaaacaac gaagtcaata 1800aaagacttag agactgttac tgttaatgga agcgattata aacggttgga agtgactgtt 1860tctatcgaca atcaaggagg agctacagga atcgctttat cattagtagg atcccaactc 1920gatttgttag aacctgtcta catcgataat attgaacttc taaattcctt tgaagcacca 1980ccagcagatt cttttcttgt tgatgatttt gaaggttatt ttggggatga cacgttgtta 2040catcgcaatt attctagcaa tggagatcca attacactat cgttaacaag tgagtttaaa 2100aataatggag aatttggatt gaagtatgat tattcgattg gctcgatggg ttatgcaggg 2160aggcaaacat cactaggacc tgtcgattgg agcggagcta atgcttttga attttggatg 2220aaacatggac aacttgaagg gaatcattta actgtacaaa ttcgaatagg tgatgttagc 2280tttgaaaaaa atcttgaatt aatggatgct catgaaggtg tagtgacaat cccgttttct 2340gaatttgctc cagctgcttg ggaaaataag cctggcgtta tcattgacga acaaaaattg 2400aaaagagtga gtcaatttgc tctttacaca ggcggggcta gacaatctgg aacaatctac 2460tttgatgatt tacgagcggt atatgatgaa agtttaccat cagttccagt tccgaaagag 2520gaggaagagg aaaaagaggt cgctcctatt taa 255340881PRTArtificial Sequencesynthetic Bsp Man4 sequence 40Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Thr Ser Ser 20 25 30 Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys Tyr 35 40 45 Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln Val 50 55 60 Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr Asn 65 70 75 80 Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala Val 85 90 95 Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp Gly 100 105 110 Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys Asn 115 120 125 Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile Thr 130 135 140 Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly Asp 145 150 155 160 Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys His 165 170 175 Ala Glu Phe Asn

Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His Glu 180 185 190 Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro Phe 195 200 205 His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr Ser 210 215 220 Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu Arg 225 230 235 240 Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly Ala 245 250 255 Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly Asp 260 265 270 Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp Asn 275 280 285 Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala Met 290 295 300 Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr Glu 305 310 315 320 Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu Asp 325 330 335 Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala Arg 340 345 350 Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn Asn 355 360 365 Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly Gly Asp His 370 375 380 Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp Tyr Thr Ala 385 390 395 400 Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly Lys Glu Tyr 405 410 415 Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser Pro Ile Thr 420 425 430 Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala Lys Val Ala 435 440 445 Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp Gly Ser Ser Leu 450 455 460 Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr Thr Gly Ser 465 470 475 480 Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp Val Ile Val 485 490 495 Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu Thr Ile Arg Leu 500 505 510 Phe Val Lys Ile Pro Glu Met Ser Leu Leu Thr Leu Thr Phe Asp Asp 515 520 525 Asp Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro Glu Asp Gly Val 530 535 540 Thr Ser Glu Ile Asp His Ala Ile Val Asp Gly Asp Gly Lys Leu Met 545 550 555 560 Phe Ser Val Gln Gly Met Ser Pro Thr Glu Thr Trp Gln Glu Leu Lys 565 570 575 Leu Glu Leu Thr Glu Leu Ser Asp Val Asn Ile Asp Ala Val Lys Lys 580 585 590 Met Lys Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu Glu Gly Ser Val 595 600 605 Gln Gly Ile Val Gln Leu Pro Pro Asp Trp Glu Thr Lys Tyr Gly Met 610 615 620 Asn Glu Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr Val Thr Val Asn 625 630 635 640 Gly Ser Asp Tyr Lys Arg Leu Glu Val Thr Val Ser Ile Asp Asn Gln 645 650 655 Gly Gly Ala Thr Gly Ile Ala Leu Ser Leu Val Gly Ser Gln Leu Asp 660 665 670 Leu Leu Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu Leu Asn Ser Phe 675 680 685 Glu Ala Pro Pro Ala Asp Ser Phe Leu Val Asp Asp Phe Glu Gly Tyr 690 695 700 Phe Gly Asp Asp Thr Leu Leu His Arg Asn Tyr Ser Ser Asn Gly Asp 705 710 715 720 Pro Ile Thr Leu Ser Leu Thr Ser Glu Phe Lys Asn Asn Gly Glu Phe 725 730 735 Gly Leu Lys Tyr Asp Tyr Ser Ile Gly Ser Met Gly Tyr Ala Gly Arg 740 745 750 Gln Thr Ser Leu Gly Pro Val Asp Trp Ser Gly Ala Asn Ala Phe Glu 755 760 765 Phe Trp Met Lys His Gly Gln Leu Glu Gly Asn His Leu Thr Val Gln 770 775 780 Ile Arg Ile Gly Asp Val Ser Phe Glu Lys Asn Leu Glu Leu Met Asp 785 790 795 800 Ala His Glu Gly Val Val Thr Ile Pro Phe Ser Glu Phe Ala Pro Ala 805 810 815 Ala Trp Glu Asn Lys Pro Gly Val Ile Ile Asp Glu Gln Lys Leu Lys 820 825 830 Arg Val Ser Gln Phe Ala Leu Tyr Thr Gly Gly Ala Arg Gln Ser Gly 835 840 845 Thr Ile Tyr Phe Asp Asp Leu Arg Ala Val Tyr Asp Glu Ser Leu Pro 850 855 860 Ser Val Pro Val Pro Lys Glu Glu Glu Glu Glu Lys Glu Val Ala Pro 865 870 875 880 Ile 41850PRTBacillus sp. 41Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala Ser Lys 1 5 10 15 Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly Ser Gln 20 25 30 Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr Leu Thr 35 40 45 Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe Asn Ala 50 55 60 Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser Leu Asp 65 70 75 80 Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser Ile Lys 85 90 95 Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly Ile Ile 100 105 110 Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro Tyr Gly 115 120 125 Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly Ser Lys 130 135 140 His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu Ala His 145 150 155 160 Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe Arg Pro 165 170 175 Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser Thr Thr 180 185 190 Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu Tyr Leu 195 200 205 Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser Pro Gly 210 215 220 Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr Pro Gly 225 230 235 240 Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn Lys Asp 245 250 255 Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp Leu Ala 260 265 270 Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala Phe Thr 275 280 285 Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn Thr Leu 290 295 300 Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu Asp Ala 305 310 315 320 Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp Pro Asn 325 330 335 Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly Gly Asp 340 345 350 His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp Tyr Thr 355 360 365 Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly Lys Glu 370 375 380 Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser Pro Ile 385 390 395 400 Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala Lys Val 405 410 415 Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp Gly Ser Ser 420 425 430 Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr Thr Gly 435 440 445 Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp Val Ile 450 455 460 Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu Thr Ile Arg 465 470 475 480 Leu Phe Val Lys Ile Pro Glu Met Ser Leu Leu Thr Leu Thr Phe Asp 485 490 495 Asp Asp Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro Glu Asp Gly 500 505 510 Val Thr Ser Glu Ile Asp His Ala Ile Val Asp Gly Asp Gly Lys Leu 515 520 525 Met Phe Ser Val Gln Gly Met Ser Pro Thr Glu Thr Trp Gln Glu Leu 530 535 540 Lys Leu Glu Leu Thr Glu Leu Ser Asp Val Asn Ile Asp Ala Val Lys 545 550 555 560 Lys Met Lys Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu Glu Gly Ser 565 570 575 Val Gln Gly Ile Val Gln Leu Pro Pro Asp Trp Glu Thr Lys Tyr Gly 580 585 590 Met Asn Glu Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr Val Thr Val 595 600 605 Asn Gly Ser Asp Tyr Lys Arg Leu Glu Val Thr Val Ser Ile Asp Asn 610 615 620 Gln Gly Gly Ala Thr Gly Ile Ala Leu Ser Leu Val Gly Ser Gln Leu 625 630 635 640 Asp Leu Leu Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu Leu Asn Ser 645 650 655 Phe Glu Ala Pro Pro Ala Asp Ser Phe Leu Val Asp Asp Phe Glu Gly 660 665 670 Tyr Phe Gly Asp Asp Thr Leu Leu His Arg Asn Tyr Ser Ser Asn Gly 675 680 685 Asp Pro Ile Thr Leu Ser Leu Thr Ser Glu Phe Lys Asn Asn Gly Glu 690 695 700 Phe Gly Leu Lys Tyr Asp Tyr Ser Ile Gly Ser Met Gly Tyr Ala Gly 705 710 715 720 Arg Gln Thr Ser Leu Gly Pro Val Asp Trp Ser Gly Ala Asn Ala Phe 725 730 735 Glu Phe Trp Met Lys His Gly Gln Leu Glu Gly Asn His Leu Thr Val 740 745 750 Gln Ile Arg Ile Gly Asp Val Ser Phe Glu Lys Asn Leu Glu Leu Met 755 760 765 Asp Ala His Glu Gly Val Val Thr Ile Pro Phe Ser Glu Phe Ala Pro 770 775 780 Ala Ala Trp Glu Asn Lys Pro Gly Val Ile Ile Asp Glu Gln Lys Leu 785 790 795 800 Lys Arg Val Ser Gln Phe Ala Leu Tyr Thr Gly Gly Ala Arg Gln Ser 805 810 815 Gly Thr Ile Tyr Phe Asp Asp Leu Arg Ala Val Tyr Asp Glu Ser Leu 820 825 830 Pro Ser Val Pro Val Pro Lys Glu Glu Glu Glu Glu Lys Glu Val Ala 835 840 845 Pro Ile 850 421155DNAArtificial Sequencesynthetic aprE-Bsp Man4 sequence 42gtgagaagca aaaaattgtg gatcagcttg ttgtttgcgt taacgttaat ctttacgatg 60gcgttcagca acatgagcgc gcaggcagct ggtaaaacta gttcacaaga agggcgtcaa 120cttaacatgg cagatgagga tgcttcaaag tatacgaagg agttatttgc ttttcttcaa 180gatgtaagtg gttcacaagt gttatttgga caacagcatg caacagatga aggattaact 240ttaacaaatc cagctccaag aacaggttcc actcaatctg aagttttcaa tgcagttggg 300gattatccag ctgtgtttgg atgggacacg aatagcctag atggtcgtga aaagcctggc 360attgcaggta atgtagaaca aagtataaaa aatacggctc agtccatgaa agtggctcat 420gatttaggag ggattattac actaagcatg cacccagata attttgtaac agggggtcct 480tatggtgata caacagggaa tgttgtaaaa gaaattcttc caggtggatc aaaacatgca 540gagtttaacg cgtggttgga caatattgct gcgcttgctc acgagctgaa agatgagaat 600ggtgaaccta ttccgatgat ttttcggcca ttccatgaac aaacaggatc ttggttttgg 660tggggagcaa gcacaacttc acccgaacaa tataaagcga tttttcgtta tacagtagaa 720tatttgcgag atgttaaagg cgtaaataat attttatatg gcttttcacc tggggcggga 780cctgctggag atgtaaatcg ctatttagaa acatatccag gggatgatta cgttgatatt 840ttcggtattg acaattatga caataaagac aatgcagggt cagaagcttg gttaagtggt 900atggtcaaag acttggcgat gattagccga ttagctgaac aaaaagaaaa agtagcggct 960tttactgagt atgggtacag tgcaaccgga attaatcgtc aagggaatac attagactgg 1020tacacacgtg tattagatgc gattgctgct gatgaagacg cacgtaaaat atcatacatg 1080ttgacatggg cgaactttgg ttggccgaat aatatgtatg ttccttatcg tgatatccac 1140aatgaattag gttaa 115543384PRTArtificial Sequencesynthetic aprE-Bsp Man4 sequence 43Val Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Gly Lys 20 25 30 Thr Ser Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala 35 40 45 Ser Lys Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly 50 55 60 Ser Gln Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr 65 70 75 80 Leu Thr Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe 85 90 95 Asn Ala Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser 100 105 110 Leu Asp Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser 115 120 125 Ile Lys Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly 130 135 140 Ile Ile Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro 145 150 155 160 Tyr Gly Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly 165 170 175 Ser Lys His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu 180 185 190 Ala His Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe 195 200 205 Arg Pro Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser 210 215 220 Thr Thr Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu 225 230 235 240 Tyr Leu Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser 245 250 255 Pro Gly Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr 260 265 270 Pro Gly Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn 275 280 285 Lys Asp Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp 290 295 300 Leu Ala Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala 305 310 315 320 Phe Thr Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn 325 330 335 Thr Leu Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu 340 345 350 Asp Ala Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp 355 360 365 Pro Asn Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly 370 375 380 441530DNAArtificial Sequencesynthetic aprE-Bsp Man4 sequence 44gtgagaagca aaaaattgtg gatcagcttg ttgtttgcgt taacgttaat ctttacgatg 60gcgttcagca acatgagcgc gcaggcagct ggtaaaacta gttcacaaga agggcgtcaa 120cttaacatgg cagatgagga tgcttcaaag tatacgaagg agttatttgc ttttcttcaa 180gatgtaagtg gttcacaagt gttatttgga caacagcatg caacagatga aggattaact 240ttaacaaatc cagctccaag aacaggttcc actcaatctg aagttttcaa tgcagttggg 300gattatccag ctgtgtttgg atgggacacg aatagcctag atggtcgtga aaagcctggc 360attgcaggta atgtagaaca aagtataaaa aatacggctc agtccatgaa agtggctcat 420gatttaggag ggattattac actaagcatg cacccagata attttgtaac agggggtcct 480tatggtgata caacagggaa tgttgtaaaa gaaattcttc caggtggatc aaaacatgca 540gagtttaacg cgtggttgga caatattgct gcgcttgctc acgagctgaa agatgagaat 600ggtgaaccta ttccgatgat ttttcggcca ttccatgaac aaacaggatc ttggttttgg 660tggggagcaa gcacaacttc acccgaacaa tataaagcga tttttcgtta tacagtagaa 720tatttgcgag atgttaaagg cgtaaataat attttatatg gcttttcacc tggggcggga 780cctgctggag atgtaaatcg ctatttagaa acatatccag gggatgatta cgttgatatt 840ttcggtattg acaattatga caataaagac aatgcagggt cagaagcttg gttaagtggt 900atggtcaaag acttggcgat gattagccga ttagctgaac aaaaagaaaa agtagcggct 960tttactgagt atgggtacag tgcaaccgga attaatcgtc aagggaatac attagactgg 1020tacacacgtg tattagatgc

gattgctgct gatgaagacg cacgtaaaat atcatacatg 1080ttgacatggg cgaactttgg ttggccgaat aatatgtatg ttccttatcg tgatatccac 1140aatgaattag gtggagacca tgagttatta ccggactttg aagctttcca tgcggatgac 1200tacacagcat ttcgagatga gataaaagga aagatatata atactggaaa ggaatatacc 1260gtttctcctc atgagccgtt tatgtatgtt atatctccga ttacaggttc tacagtgaca 1320agcgaaacgg taacaatcca agcaaaagta gcgaatgacg aacacgcaag agtcactttc 1380agggtcgatg gttctagttt ggaagaagaa atggttttca atgatgacac tttatattat 1440acaggttctt ttacaccaga tgcagcagtg aatggcggag ctgttgatgt gattgtagct 1500tattattcta gtggagaaaa agtccaataa 153045509PRTArtificial Sequencesynthetic aprE-Bsp Man4 sequence 45Val Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Gly Lys 20 25 30 Thr Ser Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala 35 40 45 Ser Lys Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly 50 55 60 Ser Gln Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr 65 70 75 80 Leu Thr Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe 85 90 95 Asn Ala Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser 100 105 110 Leu Asp Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser 115 120 125 Ile Lys Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly 130 135 140 Ile Ile Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro 145 150 155 160 Tyr Gly Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly 165 170 175 Ser Lys His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu 180 185 190 Ala His Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe 195 200 205 Arg Pro Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser 210 215 220 Thr Thr Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu 225 230 235 240 Tyr Leu Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser 245 250 255 Pro Gly Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr 260 265 270 Pro Gly Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn 275 280 285 Lys Asp Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp 290 295 300 Leu Ala Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala 305 310 315 320 Phe Thr Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn 325 330 335 Thr Leu Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu 340 345 350 Asp Ala Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp 355 360 365 Pro Asn Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly 370 375 380 Gly Asp His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp 385 390 395 400 Tyr Thr Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly 405 410 415 Lys Glu Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser 420 425 430 Pro Ile Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala 435 440 445 Lys Val Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp Gly 450 455 460 Ser Ser Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr 465 470 475 480 Thr Gly Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp 485 490 495 Val Ile Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln 500 505 462130DNAArtificial Sequencesynthetic aprE-Bsp Man4 sequence 46gtgagaagca aaaaattgtg gatcagcttg ttgtttgcgt taacgttaat ctttacgatg 60gcgttcagca acatgagcgc gcaggcagct ggtaaaacta gttcacaaga agggcgtcaa 120cttaacatgg cagatgagga tgcttcaaag tatacgaagg agttatttgc ttttcttcaa 180gatgtaagtg gttcacaagt gttatttgga caacagcatg caacagatga aggattaact 240ttaacaaatc cagctccaag aacaggttcc actcaatctg aagttttcaa tgcagttggg 300gattatccag ctgtgtttgg atgggacacg aatagcctag atggtcgtga aaagcctggc 360attgcaggta atgtagaaca aagtataaaa aatacggctc agtccatgaa agtggctcat 420gatttaggag ggattattac actaagcatg cacccagata attttgtaac agggggtcct 480tatggtgata caacagggaa tgttgtaaaa gaaattcttc caggtggatc aaaacatgca 540gagtttaacg cgtggttgga caatattgct gcgcttgctc acgagctgaa agatgagaat 600ggtgaaccta ttccgatgat ttttcggcca ttccatgaac aaacaggatc ttggttttgg 660tggggagcaa gcacaacttc acccgaacaa tataaagcga tttttcgtta tacagtagaa 720tatttgcgag atgttaaagg cgtaaataat attttatatg gcttttcacc tggggcggga 780cctgctggag atgtaaatcg ctatttagaa acatatccag gggatgatta cgttgatatt 840ttcggtattg acaattatga caataaagac aatgcagggt cagaagcttg gttaagtggt 900atggtcaaag acttggcgat gattagccga ttagctgaac aaaaagaaaa agtagcggct 960tttactgagt atgggtacag tgcaaccgga attaatcgtc aagggaatac attagactgg 1020tacacacgtg tattagatgc gattgctgct gatgaagacg cacgtaaaat atcatacatg 1080ttgacatggg cgaactttgg ttggccgaat aatatgtatg ttccttatcg tgatatccac 1140aatgaattag gtggagacca tgagttatta ccggactttg aagctttcca tgcggatgac 1200tacacagcat ttcgagatga gataaaagga aagatatata atactggaaa ggaatatacc 1260gtttctcctc atgagccgtt tatgtatgtt atatctccga ttacaggttc tacagtgaca 1320agcgaaacgg taacaatcca agcaaaagta gcgaatgacg aacacgcaag agtcactttc 1380agggtcgatg gttctagttt ggaagaagaa atggttttca atgatgacac tttatattat 1440acaggttctt ttacaccaga tgcagcagtg aatggcggag ctgttgatgt gattgtagct 1500tattattcta gtggagaaaa agtccaagaa gaaacaattc gtttatttgt aaaaattcct 1560gaaatgtctt tgttaacatt aacgtttgat gatgatataa acggaatcaa aagcaatgga 1620acatggcctg aagatggtgt aacatctgaa attgaccacg ctattgtaga tggagacggc 1680aagttgatgt tctctgttca aggaatgtca cctactgaaa catggcaaga gctcaagtta 1740gaattaacag aactatcaga tgtgaacatt gatgcggtta agaaaatgaa gtttgacgcg 1800cttatcccag caggtagtga agaaggttca gtccaaggaa tcgtacaact tccaccggat 1860tgggagacga aatatgggat gaatgaaaca acgaagtcaa taaaagactt agagactgtt 1920actgttaatg gaagcgatta taaacggttg gaagtgactg tttctatcga caatcaagga 1980ggagctacag gaatcgcttt atcattagta ggatcccaac tcgatttgtt agaacctgtc 2040tacatcgata atattgaact tctaaattcc tttgaagcac caccagcaga ttcttttctt 2100gttgatgatt ttgaaggtta ttttgggtaa 213047709PRTArtificial Sequencesynthetic aprE-Bsp Man4 sequence 47Val Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Gly Lys 20 25 30 Thr Ser Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala 35 40 45 Ser Lys Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly 50 55 60 Ser Gln Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr 65 70 75 80 Leu Thr Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe 85 90 95 Asn Ala Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser 100 105 110 Leu Asp Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser 115 120 125 Ile Lys Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly 130 135 140 Ile Ile Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro 145 150 155 160 Tyr Gly Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly 165 170 175 Ser Lys His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu 180 185 190 Ala His Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe 195 200 205 Arg Pro Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser 210 215 220 Thr Thr Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu 225 230 235 240 Tyr Leu Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser 245 250 255 Pro Gly Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr 260 265 270 Pro Gly Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn 275 280 285 Lys Asp Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp 290 295 300 Leu Ala Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala 305 310 315 320 Phe Thr Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn 325 330 335 Thr Leu Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu 340 345 350 Asp Ala Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp 355 360 365 Pro Asn Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly 370 375 380 Gly Asp His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp 385 390 395 400 Tyr Thr Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly 405 410 415 Lys Glu Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser 420 425 430 Pro Ile Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala 435 440 445 Lys Val Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp Gly 450 455 460 Ser Ser Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr 465 470 475 480 Thr Gly Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp 485 490 495 Val Ile Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu Thr 500 505 510 Ile Arg Leu Phe Val Lys Ile Pro Glu Met Ser Leu Leu Thr Leu Thr 515 520 525 Phe Asp Asp Asp Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro Glu 530 535 540 Asp Gly Val Thr Ser Glu Ile Asp His Ala Ile Val Asp Gly Asp Gly 545 550 555 560 Lys Leu Met Phe Ser Val Gln Gly Met Ser Pro Thr Glu Thr Trp Gln 565 570 575 Glu Leu Lys Leu Glu Leu Thr Glu Leu Ser Asp Val Asn Ile Asp Ala 580 585 590 Val Lys Lys Met Lys Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu Glu 595 600 605 Gly Ser Val Gln Gly Ile Val Gln Leu Pro Pro Asp Trp Glu Thr Lys 610 615 620 Tyr Gly Met Asn Glu Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr Val 625 630 635 640 Thr Val Asn Gly Ser Asp Tyr Lys Arg Leu Glu Val Thr Val Ser Ile 645 650 655 Asp Asn Gln Gly Gly Ala Thr Gly Ile Ala Leu Ser Leu Val Gly Ser 660 665 670 Gln Leu Asp Leu Leu Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu Leu 675 680 685 Asn Ser Phe Glu Ala Pro Pro Ala Asp Ser Phe Leu Val Asp Asp Phe 690 695 700 Glu Gly Tyr Phe Gly 705 482655DNAArtificial Sequencesynthetic aprE-Bsp Man4 sequence 48gtgagaagca aaaaattgtg gatcagcttg ttgtttgcgt taacgttaat ctttacgatg 60gcgttcagca acatgagcgc gcaggcagct ggtaaaacta gttcacaaga agggcgtcaa 120cttaacatgg cagatgagga tgcttcaaag tatacgaagg agttatttgc ttttcttcaa 180gatgtaagtg gttcacaagt gttatttgga caacagcatg caacagatga aggattaact 240ttaacaaatc cagctccaag aacaggttcc actcaatctg aagttttcaa tgcagttggg 300gattatccag ctgtgtttgg atgggacacg aatagcctag atggtcgtga aaagcctggc 360attgcaggta atgtagaaca aagtataaaa aatacggctc agtccatgaa agtggctcat 420gatttaggag ggattattac actaagcatg cacccagata attttgtaac agggggtcct 480tatggtgata caacagggaa tgttgtaaaa gaaattcttc caggtggatc aaaacatgca 540gagtttaacg cgtggttgga caatattgct gcgcttgctc acgagctgaa agatgagaat 600ggtgaaccta ttccgatgat ttttcggcca ttccatgaac aaacaggatc ttggttttgg 660tggggagcaa gcacaacttc acccgaacaa tataaagcga tttttcgtta tacagtagaa 720tatttgcgag atgttaaagg cgtaaataat attttatatg gcttttcacc tggggcggga 780cctgctggag atgtaaatcg ctatttagaa acatatccag gggatgatta cgttgatatt 840ttcggtattg acaattatga caataaagac aatgcagggt cagaagcttg gttaagtggt 900atggtcaaag acttggcgat gattagccga ttagctgaac aaaaagaaaa agtagcggct 960tttactgagt atgggtacag tgcaaccgga attaatcgtc aagggaatac attagactgg 1020tacacacgtg tattagatgc gattgctgct gatgaagacg cacgtaaaat atcatacatg 1080ttgacatggg cgaactttgg ttggccgaat aatatgtatg ttccttatcg tgatatccac 1140aatgaattag gtggagacca tgagttatta ccggactttg aagctttcca tgcggatgac 1200tacacagcat ttcgagatga gataaaagga aagatatata atactggaaa ggaatatacc 1260gtttctcctc atgagccgtt tatgtatgtt atatctccga ttacaggttc tacagtgaca 1320agcgaaacgg taacaatcca agcaaaagta gcgaatgacg aacacgcaag agtcactttc 1380agggtcgatg gttctagttt ggaagaagaa atggttttca atgatgacac tttatattat 1440acaggttctt ttacaccaga tgcagcagtg aatggcggag ctgttgatgt gattgtagct 1500tattattcta gtggagaaaa agtccaagaa gaaacaattc gtttatttgt aaaaattcct 1560gaaatgtctt tgttaacatt aacgtttgat gatgatataa acggaatcaa aagcaatgga 1620acatggcctg aagatggtgt aacatctgaa attgaccacg ctattgtaga tggagacggc 1680aagttgatgt tctctgttca aggaatgtca cctactgaaa catggcaaga gctcaagtta 1740gaattaacag aactatcaga tgtgaacatt gatgcggtta agaaaatgaa gtttgacgcg 1800cttatcccag caggtagtga agaaggttca gtccaaggaa tcgtacaact tccaccggat 1860tgggagacga aatatgggat gaatgaaaca acgaagtcaa taaaagactt agagactgtt 1920actgttaatg gaagcgatta taaacggttg gaagtgactg tttctatcga caatcaagga 1980ggagctacag gaatcgcttt atcattagta ggatcccaac tcgatttgtt agaacctgtc 2040tacatcgata atattgaact tctaaattcc tttgaagcac caccagcaga ttcttttctt 2100gttgatgatt ttgaaggtta ttttggggat gacacgttgt tacatcgcaa ttattctagc 2160aatggagatc caattacact atcgttaaca agtgagttta aaaataatgg agaatttgga 2220ttgaagtatg attattcgat tggctcgatg ggttatgcag ggaggcaaac atcactagga 2280cctgtcgatt ggagcggagc taatgctttt gaattttgga tgaaacatgg acaacttgaa 2340gggaatcatt taactgtaca aattcgaata ggtgatgtta gctttgaaaa aaatcttgaa 2400ttaatggatg ctcatgaagg tgtagtgaca atcccgtttt ctgaatttgc tccagctgct 2460tgggaaaata agcctggcgt tatcattgac gaacaaaaat tgaaaagagt gagtcaattt 2520gctctttaca caggcggggc tagacaatct ggaacaatct actttgatga tttacgagcg 2580gtatatgatg aaagtttacc atcagttcca gttccgaaag aggaggaaga ggaaaaagag 2640gtcgctccta tttaa 265549884PRTArtificial Sequencesynthetic aprE-Bsp Man4 sequence 49Val Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Gly Lys 20 25 30 Thr Ser Ser Gln Glu Gly Arg Gln Leu Asn Met Ala Asp Glu Asp Ala 35 40 45 Ser Lys Tyr Thr Lys Glu Leu Phe Ala Phe Leu Gln Asp Val Ser Gly 50 55 60 Ser Gln Val Leu Phe Gly Gln Gln His Ala Thr Asp Glu Gly Leu Thr 65 70 75 80 Leu Thr Asn Pro Ala Pro Arg Thr Gly Ser Thr Gln Ser Glu Val Phe 85 90 95 Asn Ala Val Gly Asp Tyr Pro Ala Val Phe Gly Trp Asp Thr Asn Ser 100 105 110 Leu Asp Gly Arg Glu Lys Pro Gly Ile Ala Gly Asn Val Glu Gln Ser 115 120 125 Ile Lys Asn Thr Ala Gln Ser Met Lys Val Ala His Asp Leu Gly Gly 130 135 140 Ile Ile Thr Leu Ser Met His Pro Asp Asn Phe Val Thr Gly Gly Pro 145 150 155 160 Tyr Gly Asp Thr Thr Gly Asn Val Val Lys Glu Ile Leu Pro Gly Gly 165 170 175 Ser Lys His Ala Glu Phe Asn Ala Trp Leu Asp Asn Ile Ala Ala Leu 180 185 190 Ala His Glu Leu Lys Asp Glu Asn Gly Glu Pro Ile Pro Met Ile Phe 195 200 205 Arg Pro Phe His Glu Gln Thr Gly Ser Trp Phe Trp Trp Gly Ala Ser 210 215

220 Thr Thr Ser Pro Glu Gln Tyr Lys Ala Ile Phe Arg Tyr Thr Val Glu 225 230 235 240 Tyr Leu Arg Asp Val Lys Gly Val Asn Asn Ile Leu Tyr Gly Phe Ser 245 250 255 Pro Gly Ala Gly Pro Ala Gly Asp Val Asn Arg Tyr Leu Glu Thr Tyr 260 265 270 Pro Gly Asp Asp Tyr Val Asp Ile Phe Gly Ile Asp Asn Tyr Asp Asn 275 280 285 Lys Asp Asn Ala Gly Ser Glu Ala Trp Leu Ser Gly Met Val Lys Asp 290 295 300 Leu Ala Met Ile Ser Arg Leu Ala Glu Gln Lys Glu Lys Val Ala Ala 305 310 315 320 Phe Thr Glu Tyr Gly Tyr Ser Ala Thr Gly Ile Asn Arg Gln Gly Asn 325 330 335 Thr Leu Asp Trp Tyr Thr Arg Val Leu Asp Ala Ile Ala Ala Asp Glu 340 345 350 Asp Ala Arg Lys Ile Ser Tyr Met Leu Thr Trp Ala Asn Phe Gly Trp 355 360 365 Pro Asn Asn Met Tyr Val Pro Tyr Arg Asp Ile His Asn Glu Leu Gly 370 375 380 Gly Asp His Glu Leu Leu Pro Asp Phe Glu Ala Phe His Ala Asp Asp 385 390 395 400 Tyr Thr Ala Phe Arg Asp Glu Ile Lys Gly Lys Ile Tyr Asn Thr Gly 405 410 415 Lys Glu Tyr Thr Val Ser Pro His Glu Pro Phe Met Tyr Val Ile Ser 420 425 430 Pro Ile Thr Gly Ser Thr Val Thr Ser Glu Thr Val Thr Ile Gln Ala 435 440 445 Lys Val Ala Asn Asp Glu His Ala Arg Val Thr Phe Arg Val Asp Gly 450 455 460 Ser Ser Leu Glu Glu Glu Met Val Phe Asn Asp Asp Thr Leu Tyr Tyr 465 470 475 480 Thr Gly Ser Phe Thr Pro Asp Ala Ala Val Asn Gly Gly Ala Val Asp 485 490 495 Val Ile Val Ala Tyr Tyr Ser Ser Gly Glu Lys Val Gln Glu Glu Thr 500 505 510 Ile Arg Leu Phe Val Lys Ile Pro Glu Met Ser Leu Leu Thr Leu Thr 515 520 525 Phe Asp Asp Asp Ile Asn Gly Ile Lys Ser Asn Gly Thr Trp Pro Glu 530 535 540 Asp Gly Val Thr Ser Glu Ile Asp His Ala Ile Val Asp Gly Asp Gly 545 550 555 560 Lys Leu Met Phe Ser Val Gln Gly Met Ser Pro Thr Glu Thr Trp Gln 565 570 575 Glu Leu Lys Leu Glu Leu Thr Glu Leu Ser Asp Val Asn Ile Asp Ala 580 585 590 Val Lys Lys Met Lys Phe Asp Ala Leu Ile Pro Ala Gly Ser Glu Glu 595 600 605 Gly Ser Val Gln Gly Ile Val Gln Leu Pro Pro Asp Trp Glu Thr Lys 610 615 620 Tyr Gly Met Asn Glu Thr Thr Lys Ser Ile Lys Asp Leu Glu Thr Val 625 630 635 640 Thr Val Asn Gly Ser Asp Tyr Lys Arg Leu Glu Val Thr Val Ser Ile 645 650 655 Asp Asn Gln Gly Gly Ala Thr Gly Ile Ala Leu Ser Leu Val Gly Ser 660 665 670 Gln Leu Asp Leu Leu Glu Pro Val Tyr Ile Asp Asn Ile Glu Leu Leu 675 680 685 Asn Ser Phe Glu Ala Pro Pro Ala Asp Ser Phe Leu Val Asp Asp Phe 690 695 700 Glu Gly Tyr Phe Gly Asp Asp Thr Leu Leu His Arg Asn Tyr Ser Ser 705 710 715 720 Asn Gly Asp Pro Ile Thr Leu Ser Leu Thr Ser Glu Phe Lys Asn Asn 725 730 735 Gly Glu Phe Gly Leu Lys Tyr Asp Tyr Ser Ile Gly Ser Met Gly Tyr 740 745 750 Ala Gly Arg Gln Thr Ser Leu Gly Pro Val Asp Trp Ser Gly Ala Asn 755 760 765 Ala Phe Glu Phe Trp Met Lys His Gly Gln Leu Glu Gly Asn His Leu 770 775 780 Thr Val Gln Ile Arg Ile Gly Asp Val Ser Phe Glu Lys Asn Leu Glu 785 790 795 800 Leu Met Asp Ala His Glu Gly Val Val Thr Ile Pro Phe Ser Glu Phe 805 810 815 Ala Pro Ala Ala Trp Glu Asn Lys Pro Gly Val Ile Ile Asp Glu Gln 820 825 830 Lys Leu Lys Arg Val Ser Gln Phe Ala Leu Tyr Thr Gly Gly Ala Arg 835 840 845 Gln Ser Gly Thr Ile Tyr Phe Asp Asp Leu Arg Ala Val Tyr Asp Glu 850 855 860 Ser Leu Pro Ser Val Pro Val Pro Lys Glu Glu Glu Glu Glu Lys Glu 865 870 875 880 Val Ala Pro Ile

Claims

1. A recombinant polypeptide comprising a catalytic domain of an endo-.beta.-mannanase, wherein the catalytic domain is at least 85% identical to the amino acid sequence of SEQ ID NO:9 or a mature form of an endo-.beta.-mannanase, wherein the mature form is at least 80% identical to the amino acid sequence of SEQ ID NO:8.

2. (canceled)

3. The recombinant polypeptide of claim 1, wherein the polypeptide has mannanase activity in the presence of detergent.

4. The recombinant polypeptide of claim 1, wherein the polypeptide has mannanase activity in the presence of a protease.

5. The recombinant polypeptide of claim 1, wherein the polypeptide retains greater than 70% mannanase activity at pH values of between 6 and 8.5.

6. The recombinant polypeptide of claim 1, wherein the polypeptide retains greater than 70% mannanase activity at a temperature range from 55.degree. C. to 65.degree. C.

7. The recombinant polypeptide of claim 1, wherein the polypeptide is capable of hydrolyzing a substrate selected from the group consisting of chocolate ice cream, guar gum, locust bean gum, and combinations thereof.

8. The recombinant polypeptide of claim 1, wherein the amino acid sequence is at least 95% identical to one of the group consisting of SEQ ID NOS:6-14 and 30-49.

9. (canceled)

10. The recombinant polypeptide of claim 1, further comprising a native or non-native signal peptide.

11. The recombinant polypeptide of claim 1, wherein the polypeptide does not further comprise a carbohydrate-binding module.

12. A detergent composition comprising the recombinant polypeptide of claim 1.

13. The detergent composition of claim 12, further comprising a surfactant.

14. The detergent composition of claim 13, wherein the surfactant is an ionic surfactant.

15. The detergent composition of claim 13, wherein the surfactant is selected from the group consisting of an anionic surfactant, a cationic surfactant, a zwitterionic surfactant, and a combination thereof.

16. The detergent composition of claim 12, further comprising an enzyme selected from the group consisting of proteases, peroxidases, cellulases, beta-glucanases, hemicellulases, lipases, acyl transferases, phospholipases, esterases, laccases, catalases, aryl esterases, amylases, alpha-amylases, glucoamylases, cutinases, pectinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenases, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, other endo-.beta.-mannanases, exo-.beta.-mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, and combinations thereof.

17. (canceled)

18. The detergent composition of claim 12, wherein the detergent is selected from the group consisting of a laundry detergent, a fabric softening detergent, a dishwashing detergent, and a hard-surface cleaning detergent.

19. The detergent composition of claim 12, wherein the detergent is in a form selected from the group consisting of a liquid, a powder, a granulated solid, and a tablet.

20. A method for hydrolyzing a mannan substrate present in a soil or stain on a surface, comprising: contacting the surface with the detergent composition of claim 12 to produce a clean surface.

21. A method of textile cleaning comprising: contacting a soiled textile with the detergent composition of claim 12 to produce a clean textile.

22-39. (canceled)

40. The detergent composition of claim 13, wherein the surfactant is a non-ionic surfactant.